Omega-hydroxylase-related fusion polypeptides with improved properties

ABSTRACT

The disclosure relates to omega-hydroxylase-related fusion polypeptides that result in improved omega-hydroxylated fatty acid derivative production when expressed in recombinant host cells. The disclosure further relates to microorganisms for expressing the omega-hydroxylase-related fusion polypeptides for the production of omega-hydroxylated fatty acid derivatives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the continuation of U.S. application Ser. No.15/319,272 filed Dec. 15, 2016, which is a U.S. National StageApplication under 35 U.S.C. § 371 of International Application No.PCT/US2015/036078, filed Jun. 16, 2015, which claims the benefit of U.S.Provisional Application No. 62/012,970, filed Jun. 16, 2014, the entiredisclosures of which are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporation by referencein its entirety. Said ASCII copy, created on Jan. 6, 2020 is named109112-0936 SL.txt and is 229,376 bytes in size.

FIELD

The disclosure relates to omega-hydroxylase-related fusion polypeptidesthat result in improved omega-hydroxylated fatty acid derivativeproduction when expressed in recombinant host cells. The disclosurefurther relates to microorganisms for expressing theomega-hydroxylase-related fusion polypeptides for the production ofomega-hydroxylated fatty acid derivatives.

BACKGROUND

Cytochrome P450 monooxygenases (P450s) are a diverse group of enzymes.They are categorized into families and subfamilies. When they share agreater or equal than forty percent amino acid identity they belong tothe same family. When they share a greater or equal than fiftyfivepercent amino acid identity they belong to the same subfamily. P450s usefatty acids as substrates and catalyze hydroxylation reactions. Bacteriahave several P450 systems involved in alkane degradation and fatty acidmodification and more than 1000 microbial P450s are known to date. Oneparticular P450 subfamily is known as cyp153A, wherein the first wascloned from Acinetobacter calcoaceticus in 2001. Since then, similarenzymes have been identified in other alkane-utilizing species such asSphingomonas sp. HXN200, Mycobacterium sp. HXN1500, and Alcanivoraxborkumensis (Van Bogaert et al. (2011) FEBS Journal 278:206-221).Several P450s from the bacterial CYP153A subfamily are alkaneomega-hydroxylases (ω-hdyroxylases, also referred to as ω-oxygenases)with high terminal regioselectivity. CYP153As have also been associatedwith the synthesis of industrially relevant omega-hydroxylated(ω-hydroxylated) aliphatic compounds, such as primary alcohols,ω-hydroxylated fatty acids and bi-functional fatty acid derivatives suchas α,ω-dicarboxylic acids and α,ω-diols (Honda Malca et al. (2012) Chem.Commun. 48:5115-5117).

SUMMARY

The present disclosure provides omega-hydroxylase-related fusionpolypeptides and variants thereof that can produce omega-hydroxylated-and bi-functional fatty acid derivatives in host cells. Morespecifically, the present disclosure provides CYP153A-reductase hybridfusion polypeptide variants that produceomega-hydroxylated-(ω-hydroxylated) and bi-functional fatty acidderivatives and compositions thereof including ω-hydroxylated fattyacids, ω-hydroxylated fatty esters, α,ω-diacids, α,ω-diesters, α,ω-diolsand chemicals derived therefrom such as macrolactones. Also provided arespecific CYP153A-reductase hybrid fusion nucleic acid and proteinsequences as well as recombinant host cells and cell cultures thatencompass such engineered CYP153A-reductase hybrid fusion polypeptidevariants. The disclosure also provides methods of using the recombinantCYP153A-reductase hybrid fusion polypeptide variant-expressing hostcells in order to make ω-hydroxylated and/or bi-functional fatty acidderivatives or compositions thereof.

One aspect of the disclosure provides a CYP153A-reductase hybrid fusionpolypeptide that catalyzes the conversion of a fatty acid to anω-hydroxylated (ω-OH) fatty acid or fatty acid derivative, wherein theCYP153A-reductase hybrid fusion polypeptide has at least about 90%, 91%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to thepolypeptide sequence of SEQ ID NO: 6. Further included are methods forexpressing the CYP153A-reductase hybrid fusion polypeptide and variantsthereof. In one aspect, the CYP153A-reductase hybrid fusion polypeptidehas at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO: 6 and expression of theCYP153A-reductase hybrid fusion polypeptide in a recombinant host cellresults in a higher titer of ω-OH fatty acids or fatty acid derivativesor compositions thereof as compared to the titer produced by expressionof a wild type CYP153A. In one aspect, the recombinant host cellproduces an ω-OH fatty acid or ω-OH fatty acid derivative or compositionthereof with a titer that is at least about 10% greater than the titerof an ω-OH fatty acid or ω-OH fatty acid derivative or compositionthereof produced by a host cell expressing a corresponding wild typeCYP153A, when cultured in medium containing a carbon source underconditions effective to express the CYP153A-reductase hybrid fusionpolypeptide. In another aspect, the ω-OH fatty acid or ω-OH fatty acidderivative or composition thereof is produced extracellularly.

Another aspect of the present disclosure provides a CYP153A-reductasehybrid fusion polypeptide variant having at least about 90%, 91%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6 andhaving at least one mutation at an amino acid position includingposition 796, 141, 231, 27, 82, 178, 309, 407, 415, 516 and/or 666,wherein the CYP153A-reductase hybrid fusion polypeptide variantcatalyzes the conversion of a fatty acid to an ω-OH fatty acid. TheCYP153A-reductase hybrid fusion polypeptide variant has a mutation atany one or more of the following positions, including position A796Vwhere alanine (A) is substituted with (i.e., replaced with) valine (V);position V141I where valine is substituted with isoleucine (I); positionV141Q where valine (V) is substituted with glutamine (Q); position V141Gwhere valine (V) is substituted with glycine (G); position V141M wherevaline (V) is substituted with methionine (M); position V141L wherevaline (V) is substituted with leucine (L); position V141T where valine(V) substituted with threonine (T); position A231T where alanine (A) issubstituted with threonine (T); position R27L where arginine (R) issubstituted with lysine (L); position R82D where arginine (R) issubstituted with aspartic acid (D); position R178N where arginine (R) issubstituted with asparagine (N); position N309R where asparagine (N) issubstituted with arginine (R); position N407A where asparagine (N) issubstituted with alanine (A); position V415R where valine (V) issubstituted with arginine (R); position T516V where threonine (T) issubstituted with valine (V); position P666A where proline (P) issubstituted with alanine (A); and position P666D where proline (P) issubstituted with aspartic acid (D). Examples of CYP153A-reductase hybridfusion polypeptide variants include SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46. In one embodiment,the CYP153A-reductase hybrid fusion polypeptide variant is a hybridcyp153A-RedRhF-type fusion protein variant. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant in a recombinanthost cell results in a higher titer of an the ω-OH fatty acid or ω-OHfatty acid derivative or composition thereof as compared to the titer ofan the ω-OH fatty acid or ω-OH fatty acid derivative or compositionthereof produced by expression of a CYP153A-reductase hybrid fusionpolypeptide in a corresponding host cell. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant has a mutation atamino acid position 796, including A796V. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant has a mutation atamino acid position 231, including A231T. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant has a mutation atamino acid position 141, including V141I and/or V141T. Herein, theexpression of the CYP153A-reductase hybrid fusion polypeptide variantwith mutations A796V, V141I, V141T and/or A231T in a recombinant hostcell result in a higher titer of an ω-OH C₁₂ or C₁₆ fatty acid,respectively, as compared to a titer of an ω-OH C₁₂ or C₁₆ fatty acidproduced by expression of a CYP153A-reductase hybrid fusion polypeptide.

The disclosure further contemplates a cell culture with a recombinanthost cell expressing a CYP153A-reductase hybrid fusion polypeptidevariant as described above (supra). The ω-OH fatty acid or fatty acidderivative or composition thereof may include one or more of a C₆, C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉ and a C₂₀ ω-OHfatty acid or fatty acid derivative. The ω-OH fatty acid or fatty acidderivative or composition thereof may include a saturated or unsaturatedω-OH fatty acid or fatty acid derivative. In another embodiment, theω-OH fatty acid or fatty acid derivative or composition thereof mayinclude one or more of a C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1),C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) anda C_(20:1) unsaturated ω-OH fatty acid or fatty acid derivative. Inanother embodiment, the ω-OH fatty acid or fatty acid derivative orcomposition thereof may include an ω-OH C₁₂ and/or C₁₆ and/or C_(16:1)fatty acid or fatty acid derivative.

Another aspect of the disclosure provides a method of producing an ω-OHfatty acid or fatty acid derivative or composition thereof having anincrease in titer, including culturing the host cell expressing theCYP153A-reductase hybrid fusion polypeptide variant (supra) with acarbon source; and harvesting an ω-OH fatty acid or an ω-OH fatty acidderivative. In one aspect, the ω-OH fatty acid or fatty acid derivativeis at least about 20% to 30% greater than the titer of an ω-OH fattyacid or fatty acid derivative produced by a CYP153A-reductase hybridfusion polypeptide-expressing host cell. In another aspect, the ω-OHfatty acid or fatty acid derivative or composition thereof is producedat a titer of about 15 g/L to about 25 g/L from a carbon source.

Another aspect of the disclosure provides a CYP153A-reductase hybridfusion polypeptide variant with at least about 90%, 91%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32 having amutation at V141I and A231T, wherein the CYP153A-reductase hybrid fusionpolypeptide variant catalyzes the conversion of a fatty acid to an ω-OHC₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀,C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1),C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fattyacid or fatty acid derivative or composition thereof. Another aspect ofthe disclosure provides a CYP153A-reductase hybrid fusion polypeptidevariant with at least about 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to SEQ ID NO: 34 having a mutation at R27L,R82D, V141M, R178N and N407A, wherein the CYP153A-reductase hybridfusion polypeptide variant catalyzes the conversion of a fatty acid toan ω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈,C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1),C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/orC_(20:1) fatty acid or fatty acid derivative or composition thereof.Another aspect of the disclosure provides a CYP153A-reductase hybridfusion polypeptide variant with at least about 90%, 91%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36 having amutation at P666A, wherein the CYP153A-reductase hybrid fusionpolypeptide variant catalyzes the conversion of a fatty acid to an ω-OHC₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀,C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1),C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fattyacid or fatty acid derivative or composition thereof. Another aspect ofthe disclosure provides a CYP153A-reductase hybrid fusion polypeptidevariant with at least about 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to SEQ ID NO: 38 having a mutation at A796V,wherein the CYP153A-reductase hybrid fusion polypeptide variantcatalyzes the conversion of a fatty acid to an ω-OH C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1),C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1),C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fatty acid or fatty acidderivative or composition thereof. Another aspect of the disclosureprovides a CYP153A-reductase hybrid fusion polypeptide variant with atleast about 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO: 40 having a mutation at A796V, P666D and T516V,wherein the CYP153A-reductase hybrid fusion polypeptide variantcatalyzes the conversion of a fatty acid to an ω-OH C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1),C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1),C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fatty acid or fatty acidderivative or composition thereof. Another aspect of the disclosureprovides a CYP153A-reductase hybrid fusion polypeptide variant with atleast about 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO: 42 having a mutation at V141I, A231T and A796V,wherein the CYP153A-reductase hybrid fusion polypeptide variantcatalyzes the conversion of a fatty acid to an ω-OH C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1),C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1),C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fatty acid or fatty acidderivative or composition thereof. Another aspect of the disclosureprovides a CYP153A-reductase hybrid fusion polypeptide variant with atleast about 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO: 44 having a mutation at R27L, R82D, V141M, R178N,N407A and A796V, wherein the CYP153A-reductase hybrid fusion polypeptidevariant catalyzes the conversion of a fatty acid to an ω-OH C₆, C₇, C₈,C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1),C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1), C_(15:1),C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fatty acid orfatty acid derivative or composition thereof. Another aspect of thedisclosure provides a CYP153A-reductase hybrid fusion polypeptidevariant with at least about 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity to SEQ ID NO: 46 having a mutation at V141T,A231T and A796V, wherein the CYP153A-reductase hybrid fusion polypeptidevariant catalyzes the conversion of a fatty acid to an ω-OH C₆, C₇, C₈,C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1),C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1), C_(15:1),C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fatty acid orfatty acid derivative or composition thereof.

The disclosure further contemplates a recombinant host cell expressingthe CYP153A-reductase hybrid fusion polypeptide variant as discussedabove (supra). In one embodiment, the recombinant host cell expresses aCYP153A-reductase hybrid fusion polypeptide variant as discussed above(supra) and a thioesterase polypeptide of EC 3.1.2.- or EC 3.1.1.5 or EC3.1.2.14, wherein the recombinant host cell produces an ω-OH fatty acidor a composition thereof with a titer that is at least 10% greater, atleast 15% greater, at least 20% greater, at least 25% greater, or atleast 30% greater than the titer of an ω-OH fatty acid or compositionthereof produced by a host cell expressing a correspondingCYP153A-reductase hybrid fusion polypeptide, when cultured in mediumcontaining a carbon source under conditions effective to express theCYP153A-reductase hybrid fusion polypeptide variant. In one embodiment,the ω-OH fatty acid or composition thereof can be produced at a titer ofabout 15 g/L to about 25 g/L. In another embodiment, the ω-OH fatty acidor composition thereof is produced extracellularly.

In one aspect, the disclosure encompasses a recombinant microorganismfor producing an ω-OH fatty acid or fatty acid derivative in vivo whengrown in a fermentation broth in a presence of a carbon source from arenewable feedstock, the microorganism having a pathway engineered toexpress at least two nucleic acid sequences encoding a polypeptideincluding a thioesterase of EC 3.1.2.- or EC 3.1.1.5 or 3.1.2.14; and aCYP153A-reductase hybrid fusion polypeptide variant, wherein theCYP153A-reductase hybrid fusion polypeptide variant has at least 90%,91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to anyone of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 SEQ ID NO: 24, SEQID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46. In oneembodiment, the CYP153A-reductase hybrid fusion polypeptide variant is aself-sufficient CYP153A-RedRhF hybrid fusion protein variant.

Another aspect of the present disclosure provides a cell cultureincluding the recombinant host cell as discussed above (supra), whereinthe cell culture produces an ω-OH fatty acid or composition thereof. Inone embodiment, the cell culture produces an ω-OH fatty acid includingone or more of a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1),C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/orC_(20:1) fatty acid or fatty acid derivative or composition thereof. Inone embodiment, the cell culture produces an unsaturated ω-OH C_(16:1)fatty acid or composition thereof. In another embodiment, the cellculture produces a saturated ω-OH C₁₆ fatty acid or composition thereof.In one embodiment, the cell culture produces an unsaturated ω-OHC_(12:1) fatty acid or composition thereof. In another embodiment, thecell culture produces a saturated ω-OH C₁₂ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(14:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₁₄ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(18:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₁₈ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(10:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₁₀ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(8:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₈ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(20:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₂₀ fatty acid or compositionthereof. In yet another embodiment, additional saturated or unsaturatedω-OH fatty acids or compositions thereof are produced by the recombinanthost cell.

Still another aspect of the present disclosure provides a method ofproducing an ω-OH fatty acid having an increase in titer, includingculturing the host cell (supra) with a carbon source; and harvesting anω-OH fatty acid or composition thereof. The method contemplatesharvesting an ω-OH fatty acid that is a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂,C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1),C_(11:1), C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1),C_(18:1), C_(19:1) and/or C_(20:1) fatty acid or fatty acid derivativeor composition thereof. In one embodiment, the harvested ω-OH fatty acidis an unsaturated ω-OH C_(16:1) fatty acid or composition thereof. Inanother embodiment, the harvested ω-OH fatty acid is a saturated ω-OHC₁₆ fatty acid or composition thereof. In one embodiment, the harvestedω-OH fatty acid is an unsaturated ω-OH C_(12:1) fatty acid orcomposition thereof. In another embodiment, the harvested ω-OH fattyacid is a saturated ω-OH C₁₂ fatty acid or composition thereof. In oneembodiment, the harvested ω-OH fatty acid is an unsaturated ω-OHC_(14:1) fatty acid or composition thereof. In another embodiment, theharvested ω-OH fatty acid is a saturated ω-OH C₁₄ fatty acid orcomposition thereof. In one embodiment, the harvested ω-OH fatty acid isan unsaturated ω-OH C_(18:1) fatty acid or composition thereof. Inanother embodiment, the harvested ω-OH fatty acid is saturated ω-OH C₁₈fatty acid or composition thereof. In one embodiment, the harvested ω-OHfatty acid is an unsaturated ω-OH C_(10:1) fatty acid or compositionthereof. In another embodiment, the harvested ω-OH fatty acid is asaturated ω-OH C₁₀ fatty acid or composition thereof. In one embodiment,the harvested ω-OH fatty acid is an unsaturated ω-OH C_(8:1) fatty acidor composition thereof. In another embodiment, the harvested ω-OH fattyacid is a saturated ω-OH C₈ fatty acid or composition thereof. In oneembodiment, the harvested ω-OH fatty acid is an unsaturated ω-OHC_(20:1) fatty acid or composition thereof. In another embodiment, theharvested ω-OH fatty acid is a saturated ω-OH C₂₀ fatty acid orcomposition thereof. In yet another embodiment, additional saturated orunsaturated ω-OH fatty acids or compositions thereof are produced by themethod described herein.

Another aspect of the present disclosure provides a CYP153A-reductasehybrid fusion polypeptide variant having at least about 90%, 91%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38 andhaving at least one mutation at an amino acid position includingposition 9, 10, 11, 12, 13, 14, 27, 28, 56, 61, 111, 119, 140, 149, 154,157, 162, 164, 204, 231, 233, 244, 254, 271, 273, 302, 309, 327, 407,413, 477, 480, 481, 527, 544, 546, 557, 567, 591, 648, 649, 703, 706,707, 708, 709, 710, 719, 720, 736, 741, 745, 747, 749, 757, 770, 771,784, wherein the CYP153A-reductase hybrid fusion polypeptide variantcatalyzes the conversion of a fatty acid to an ω-OH fatty acid orcomposition thereof. The CYP153A-reductase hybrid fusion polypeptidevariant has a mutation at any one or more of the following positions,including position D9N where aspartate (D) is substituted with (i.e.,replaced with) asparagine (N); position D9K where aspartate (D) issubstituted with lysine (K); position D10Y where aspartic acid (D) issubstituted with tyrosine (Y); position I11L where isoleucine (I) issubstituted with leucine (L); position Q12W where glutamine (Q) issubstituted with tryptophan (W); position Q12R where glutamine (Q) issubstituted with arginine (R); position Q12T where glutamine (Q) issubstituted with threonine (T); position S13K where serine (S) issubstituted with lysine (K); position R14F where arginine (R) issubstituted with phenylalanine (F); position R27L where arginine (R)substituted with leucine (L); position Q28M where glutamine (Q) issubstituted with methionine (M); position Q28T where glutamine (Q) issubstituted with threonine (T); position P56Q where proline (P) issubstituted with glutamine (Q); position N61L where asparagine (N) issubstituted with leucine (L); position F111A where phenylalanine (F) issubstituted with alanine (A); position K119R where lysine (K) issubstituted with arginine (R); position S140N where serine (S) issubstituted with asparagine (N); position P149G where proline (P) issubstituted with glycine (G); position P149R where proline (P) issubstituted with arginine (R); position V154G where valine (V) issubstituted with glycine (G); position S157R where serine (S) issubstituted with arginine (R); position V162C where valine (V) issubstituted with cysteine (C); position A164N where alanine (A) issubstituted with asparagine (N); position G204V where glycine (G) issubstituted with valine (V); position A231W where alanine (A) issubstituted with tryptophan (W); position A231Y where alanine (A) issubstituted with tyrosine (Y); position A231V where alanine (A) issubstituted with valine (V); position S233L where serine (S) issubstituted with leucine (L); position S233V where serine (S) issubstituted with valine (V); position A244R where alanine (A) issubstituted with arginine (R); position R254G where arginine (R) issubstituted with glycine (G); position E271D where glutamate (E) issubstituted with aspartate (D); position P273M where proline (P) issubstituted with methionine (M); position T302M where threonine (T) issubstituted with methionine (M); position N309S where asparagine (N) issubstituted with serine (S); position P327D where proline (P) issubstituted with aspartate (D); position N407G where asparagine (N) issubstituted with glycine (G); position Y413R where tyrosine (Y) issubstituted with arginine (R); position P477G where proline (P) issubstituted with glycine (G); position I480G where isoleucine (I) issubstituted with glycine (G); position G481I where glycine (G) issubstituted with isoleucine (I); position D527E where aspartate (D) issubstituted with glutamate (E); position D544N where aspartate (D) issubstituted with asparagine (N); position P546G where proline (P) issubstituted with glycine (G); position E557R where glutamate (E) issubstituted with arginine (R); position E557W where glutamate (E) issubstituted with tryptophan (W); position E567S where glutamate (E) issubstituted with serine (S); position E591Q where glutamate (E) issubstituted with glutamine (Q); position V648L where valine (V) issubstituted with leucine (L); position S649I where serine (S) issubstituted with isoleucine (I); position L703G where leucine (L) issubstituted with glycine (G); position L706E where leucine (L) issubstituted with glutamate (E); position L706S where leucine (L) issubstituted with serine (S); position L706H where leucine (L) issubstituted with histidine (H); position D707E where aspartate (D) issubstituted with glutamate (E); position P708S where proline (P) issubstituted with serine (S); position D709L where aspartate (D) issubstituted with leucine (L); position V710C where valine (V) issubstituted with cysteine (C); position V710R where valine (V) issubstituted with arginine (R); position V710Q where valine (V) issubstituted with glutamine (Q); position R719W where arginine (R) issubstituted with tryptophan (W); position D720V where aspartate (D) issubstituted with valine (V); position A736V where alanine (A) issubstituted with valine (V); position N741G where asparagine (N) issubstituted with glycine (G); position P745K where proline (P) issubstituted with lysine (K); position P745R where proline (P) issubstituted with arginine (R); position D747N where aspartate (D) issubstituted with asparagine (N); position E749L where glutamate (E) issubstituted with leucine (L); position E749M where glutamate (E) issubstituted with methionine (M); position E757A where glutamate (E) issubstituted with alanine (A); position T770G where threonine (T) issubstituted with glycine (G); position V771F where valine (V) issubstituted with phenylalanine (F); and position M784I where methionine(M) is substituted with isoleucine (I). In one embodiment, theCYP153A-reductase hybrid fusion polypeptide variant is a hybridcyp153A-RedRhF-type fusion protein variant. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant in a recombinanthost cell results in a higher titer of an ω-OH fatty acid as compared tothe titer of an ω-OH fatty acid produced by expression of aCYP153A-reductase hybrid fusion polypeptide in a corresponding hostcell. In another embodiment, the ω-OH fatty acid is an ω-OH fatty acidcomposition.

Another aspect of the present disclosure provides a CYP153A-reductasehybrid fusion polypeptide variant having at least about 90%, 91%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38 andhaving at least one mutation at an amino acid position includingposition 747, 12, 327, 14, 61, 28, 13, 771, 119, 10, 11, 28, 745, 9,770, 413, 784, 749, 233, 757, and 703, wherein the CYP153A-reductasehybrid fusion polypeptide variant catalyzes the conversion of a fattyacid to an ω-OH fatty acid. The CYP153A-reductase hybrid fusionpolypeptide variant has a mutation at any one or more of the followingpositions, including position D747N where aspartate (D) is substitutedwith asparagine (N); position Q12W where glutamine (Q) is substitutedwith tryptophan (W); position Q12R where glutamine (Q) is substitutedwith arginine (R); position Q12T where glutamine (Q) is substituted withthreonine (T); position P327D where proline (P) is substituted withaspartate (D); position R14F where arginine (R) is substituted withphenylalanine (F); position N61L where asparagine (N) is substitutedwith leucine (L); position Q28M where glutamine (Q) is substituted withmethionine (M); position S13K where serine (S) is substituted withlysine (K); position V771F where valine (V) is substituted withphenylalanine (F); position K119R where lysine (K) is substituted witharginine (R); position D10Y where aspartic acid (D) is substituted withtyrosine (Y); position I11L where isoleucine (I) is substituted withleucine (L); position Q28T where glutamine (Q) is substituted withthreonine (T); position P745R where proline (P) is substituted witharginine (R); position D9N where aspartate (D) is substituted withasparagine (N); position D9K where aspartate (D) is substituted withlysine (K); position T770G where threonine (T) is substituted withglycine (G); position Y413R where tyrosine (Y) is substituted witharginine (R); position M784I where methionine (M) is substituted withisoleucine (I); position E749L where glutamate (E) is substituted withleucine (L); position S233L where serine (S) is substituted with leucine(L); position E757A where glutamate (E) is substituted with alanine (A);and position L703G where leucine (L) is substituted with glycine (G). Inone embodiment, the CYP153A-reductase hybrid fusion polypeptide variantis a hybrid cyp153A-RedRhF-type fusion protein variant. In anotherembodiment, the CYP153A-reductase hybrid fusion polypeptide variant in arecombinant host cell results in a higher titer of an ω-OH fatty acid ascompared to the titer of an ω-OH fatty acid produced by expression of aCYP153A-reductase hybrid fusion polypeptide in a corresponding hostcell. In another embodiment, the CYP153A-reductase hybrid fusionpolypeptide variants (and corresponding polynucleotide sequences)include SEQ ID NOS: 47-96. In another embodiment, the ω-OH fatty acid isan ω-OH fatty acid composition.

In one aspect, the disclosure encompasses a recombinant microorganism orrecombinant host cell for producing an ω-OH fatty acid or ω-OH fattyacid derivative in vivo when grown in a fermentation broth in a presenceof a carbon source from a renewable feedstock, the microorganism havinga pathway engineered to express at least two nucleic acid sequencesencoding a polypeptide including a thioesterase of EC 3.1.2.- or EC3.1.1.5 or 3.1.2.14; and a CYP153A-reductase hybrid fusion polypeptidevariant, wherein the CYP153A-reductase hybrid fusion polypeptide varianthas at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to any one of SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO:52, SEQ ID NO: 54, SEQ ID NO: 56 SEQ ID NO: 58, SEQ ID NO: 60 SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ IDNO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90,SEQ ID NO: 92, SEQ ID NO: 94, and SEQ ID NO: 96. In one embodiment, theCYP153A-reductase hybrid fusion polypeptide variant is a self-sufficientCYP153A-RedRhF hybrid fusion protein variant.

Another aspect of the present disclosure provides a cell cultureincluding the recombinant host cell as discussed above (supra), whereinthe cell culture produces an ω-OH fatty acid or composition thereof. Inone embodiment, the cell culture produces an ω-OH fatty acid includingone or more of a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1),C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/orC_(20:1) fatty acid or fatty acid derivative or composition thereof. Inone embodiment, the cell culture produces an unsaturated ω-OH C_(16:1)fatty acid or composition thereof. In another embodiment, the cellculture produces a saturated ω-OH C₁₆ fatty acid or composition thereof.In one embodiment, the cell culture produces an unsaturated ω-OHC_(12:1) fatty acid or composition thereof. In another embodiment, thecell culture produces a saturated ω-OH C₁₂ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(14:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₁₄ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(18:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₁₈ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(10:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₁₀ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(8:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₈ fatty acid or compositionthereof. In one embodiment, the cell culture produces an unsaturatedω-OH C_(10:1) fatty acid or composition thereof. In another embodiment,the cell culture produces a saturated ω-OH C₂₀ fatty acid or compositionthereof. In yet another embodiment, additional saturated or unsaturatedω-OH fatty acids or compositions thereof are produced by the recombinanthost cell.

Still another aspect of the present disclosure provides a method ofproducing an ω-OH fatty acid having an increase in titer, includingculturing the host cell (supra) with a carbon source; and harvesting anω-OH fatty acid or composition thereof. In particular, the methodencompasses producing a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅,C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1),C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1)and/or C_(20:1) fatty acid or fatty acid derivative or compositionthereof. In one embodiment, the harvested ω-OH fatty acid is anunsaturated ω-OH C_(16:1) fatty acid or composition thereof. In anotherembodiment, the harvested ω-OH fatty acid is a saturated ω-OH C₁₆ fattyacid or composition thereof. In one embodiment, the harvested ω-OH fattyacid is an unsaturated ω-OH C_(12:1) fatty acid or composition thereof.In another embodiment, the harvested ω-OH fatty acid is a saturated ω-OHC₁₂ fatty acid or composition thereof. In one embodiment, the harvestedω-OH fatty acid is an unsaturated ω-OH C_(14:1) fatty acid orcomposition thereof. In another embodiment, the harvested ω-OH fattyacid is a saturated ω-OH C₁₄ fatty acid or composition thereof. In oneembodiment, the harvested ω-OH fatty acid is an unsaturated ω-OHC_(18:1) fatty acid or composition thereof. In another embodiment, theharvested ω-OH fatty acid is saturated ω-OH C₁₈ fatty acid orcomposition thereof. In one embodiment, the harvested ω-OH fatty acid isan unsaturated ω-OH C_(10:1) fatty acid or composition thereof. Inanother embodiment, the harvested ω-OH fatty acid is a saturated ω-OHC₁₀ fatty acid or composition thereof. In one embodiment, the harvestedω-OH fatty acid is an unsaturated ω-OH C_(8:1) fatty acid or compositionthereof. In another embodiment, the harvested ω-OH fatty acid is asaturated ω-OH C₈ fatty acid or composition thereof. In one embodiment,the harvested ω-OH fatty acid is an unsaturated ω-OH C_(20:1) fatty acidor composition thereof. In another embodiment, the harvested ω-OH fattyacid is a saturated ω-OH C₂₀ fatty acid or composition thereof. In oneembodiment, the harvested ω-OH fatty acid is an unsaturated ω-OHC_(22:1) fatty acid or composition thereof. In another embodiment, theharvested ω-OH fatty acid is a saturated ω-OH C₂₂ fatty acid orcomposition thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is best understood when read in conjunction withthe accompanying figures, which serve to illustrate some preferredembodiments. It is understood, however, that the disclosure is notlimited to the specific embodiments disclosed in the figures.

FIG. 1 is a schematic overview of an exemplary biosynthetic pathway forthe production of ω-hydroxylated fatty acid derivatives such as, forexample, ω-hydroxylated C₁₂ fatty acids (ω-OH C₁₂ FFA) and/orω-hydroxylated C_(16:1) fatty acids (ω-OH C_(16:1) FFA) as a result ofexpressing the CYP153A-reductase hybrid fusion polypeptide variant and athioesterase polypeptide in a recombinant microorganism. FAB refers tofatty acid biosynthesis in the microorganism; fatB1 refers to amedium-chain acyl-ACP thioesterase from Umbellularia californica(California bay); and fatA3 refers to a long-chain acyl-ACP thioesterasefrom Arabidopsis thaliana.

FIG. 2 provides an example of the production of ω-hydroxylated fattyacids as a result of expression of a CYP153A-reductase hybrid fusionpolypeptide variant. In order to illustrate the production ofω-hydroxylated (ω-OH) fatty acids through variants a site saturationmutagenesis was employed. The depicted graph shows the best hits from asite saturation mutagenesis of the amino acid position 141 and 309 ofcyp153A(G307A, A796V)-Red450RhF. The figure refers to total fatty acidspecies (total FAS) (see dark-gray bar); to ω-hydroxy hexadecenoic acid(ω-OH C_(16:1)) (see light-gray bar); and percent ω-hydroxy fatty acids(% ω-OH FFA) (see arrow).

DETAILED DESCRIPTION General Overview

One way of eliminating our dependency on petrochemicals is to producefatty acid derivatives such as ω-OH fatty acid derivatives throughenvironmentally friendly microorganisms that serve as miniatureproduction hosts. Such cellular hosts (i.e., recombinant host cells ormicroorganisms) are engineered to produce ω-OH fatty acid derivativesand bi-functional fatty acid derivatives from renewable sources such asrenewable feedstock (e.g., fermentable carbohydrates, biomass,cellulose, glycerol, CO, CO₂, etc.). These ω-OH fatty acid derivativesare the raw materials for industrial products including specialtychemical, polymers and fragrances.

The present disclosure relates to ω-hydroxylase-related fusionpolypeptides including CYP153A-reductase hybrid fusion polypeptides andvariants thereof that result in a high titer, yield and/or productivityof ω-OH fatty acid derivative compositions when expressed in recombinanthost cells. Herein, enhanced ω-OH fatty acid derivative biosynthesis isaccomplished by transforming host cells such that they express aCYP153A-reductase hybrid fusion polypeptide or variant thereof, whichcatalyzes the reaction of a fatty acid to an ω-OH fatty acid such as,for example, an ω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆,C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1),C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1)and/or C_(20:1) fatty acid or fatty acid derivative. The disclosureencompasses the recombinant host cells or production strains thatexpress the CYP153A-reductase hybrid fusion polypeptides and variantsthereof. In one aspect, the disclosure relates to the P450 subfamilycyp153A.

Definitions

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a hostcell” includes two or more such host cells, reference to “a fatty ester”includes one or more fatty esters, or mixtures of esters, reference to“a nucleic acid sequence” includes one or more nucleic acid sequences,reference to “an enzyme” includes one or more enzymes, and the like.

The term “enzyme classification (EC) number” refers to a number thatdenotes a specific enzymatic activity. EC numbers classify enzymesaccording to the reaction they catalyze under a system of enzymenomenclature. EC numbers specify enzyme-catalyzed reactions. Forexample, if different enzymes from different organisms catalyze the samereaction, then they have the same EC number. In addition, differentprotein folds can catalyze an identical reaction and therefore would beassigned an identical EC number (e.g., non-homologous isofunctionalenzymes, or NISE). EC numbers are established by the nomenclaturecommittee of the international union of biochemistry and molecularbiology (IUBMB), a description of which is available on the IUBMB enzymenomenclature website on the world wide web. For example, the cytochromeP450 monooxygenase (P450) enzymatic activity, including theω-hydroxylase or ω-oxygenase enzymatic activity is classified under EC1.14.15.3. The functionality of enzymes that fall under the P450 enzymefamily is conserved in most prokaryotes from one species to the next.Thus, different microbial species can carry out the same enzymaticactivity that is classified under EC 1.14.15.3. An example of anenzymatic activity that is characterized by EC 1.14.15.3 is theenzymatic activity of a CYP153A-reductase hybrid fusion polypeptide orvariant thereof as discussed herein (supra).

The terms “omega-hydroxylated fatty acid” or “ω-hydroxylated fatty acid”or “ω-hydroxy fatty acid” or “ω-hydroxyl fatty acid” or “ω-OH fattyacid” or “(DOH fatty acid” are used interchangeably herein and refer toa fatty acid that originates from fatty acid metabolism and has at leastone OH group at the omega (ω) position. Examples of such ω-hydroxylatedfatty acids are C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1),C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/orC_(20:1) fatty acids. In one embodiment, such ω-hydroxylated fatty acidsare ω-OH C_(8:0) fatty acids, ω-OH C_(10:0) fatty acids, ω-OH C_(12:0)fatty acids, ω-OH C_(14:0) fatty acids, ω-OH C_(16:0) fatty acids, ω-OHC_(18:0) fatty acids, ω-OH C_(20:0) fatty acids, ω-OH C_(8:1) fattyacids, ω-OH C_(10:1) fatty acids, ω-OH C_(12:1) fatty acids, ω-OHC_(14:1) fatty acids, ω-OH C_(16:1) fatty acids, ω-OH C_(18:1) fattyacids, ω-OH C_(20:1) fatty acids, and the like. In a microorganism, theω-hydroxylated fatty acid can be used to produce ω-hydroxylated fattyacid derivatives such as ω-hydroxylated fatty esters as well asbi-functional fatty acid derivatives including α,ω-diacids,α,ω-diesters, and α,ω-diols. In that sense, the terms“omega-hydroxylated fatty acid derivative” and “ω-hydroxylated fattyacid derivative” and “ω-hydroxy fatty acid derivative” and “ω-hydroxylfatty acid derivative” and “α,ω-bifunctional fatty acid derivative” and“ω-OH fatty acid derivative” refer to a chemical entity that originatedfrom fatty acid metabolism and that has at least one OH group at theomega position or is derived from an intermediate that has at least oneOH group at the omega position. Herein, the “omega (ω) position” refersto the terminal carbon atom of a fatty acid derivative at the oppositeend in regard to its primary functional group. Such ω-hydroxylated fattyacid derivatives include, but are not limited to, α,ω-diacids;α,ω-diesters; α,ω-diols and chemicals derived thereof (e.g.,macrolactones).

An “ω-hydroxylated fatty acid composition” or “ω-OH fatty acidcomposition” as referred to herein is produced by a recombinant hostcell and typically includes a mixture of certain types of ω-hydroxylatedfatty acids with various chain lengths and/or saturation and/orbranching characteristics. Similarly, an “ω-hydroxylated fatty acidderivative composition” is produced by a recombinant host cell andtypically comprises a mixture of certain types of ω-hydroxylated fattyacid derivatives with various chain lengths and/or saturation and/orbranching characteristics (e.g., ω-hydroxylated fatty acids with variouschain lengths and/or saturation and/or branching characteristics;ω-hydroxylated fatty esters with various chain lengths and/or saturationand/or branching characteristics; α,ω-diacids of various chain lengthand/or saturation and/or branching characteristics; α,ω-diesters ofvarious chain length and/or saturation and/or branching characteristics;α,ω-diols of various chain length and/or saturation and/or branchingcharacteristics; and the like). In some cases, the ω-OH fatty acidderivative composition includes mostly one type of ω-OH fatty acidderivative such as, for example, 1,12-dodecenediol, or1,14-tetradecanediol, or 16-hydroxy hexadecanoic acid methyl ester, or16-hydroxy hexadecenoic acid, or 15-hydroxy pentadecanoic acid, or15-hydroxy pentadecenoic acid, or 18-hydroxy octacecenoic acid, or themethyl esters of any of these fatty acid derivatives, or others. Instill other cases, the ω-OH fatty acid derivative composition comprisesa mixture of more than one type of ω-OH fatty acid derivative in orderto provide a specifically designed composition (e.g., about 20%12-hydroxy dodecanoic acid and about 80% 1,14-14-hydroxy tetradecanoicacid in the same composition would provide such an example).

The term “accession number” or “NCBI accession number” or “GenBankaccession number” refers to a number that denotes a specific nucleicacid sequence. Sequence accession numbers that are discussed in thisdescription were obtained from databases provided by the NCBI (NationalCenter for Biotechnology Information) maintained by the NationalInstitutes of Health, U.S.A., and from the UniProt Knowledgebase(UniProtKB) and Swiss-Prot databases provided by the Swiss Institute ofBioinformatics (also referred to as UniProtKB accession number).

As used herein, the term “nucleotide” refers to a monomeric unit of apolynucleotide that consists of a heterocyclic base, a sugar, and one ormore phosphate groups. The naturally occurring bases (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typicallyderivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleic acids are typically linked viaphosphate bonds to form nucleic acids or polynucleotides, though manyother linkages are known in the art (e.g., phosphorothioates,boranophosphates, and the like).

The term “polynucleotide” refers to a polymer of ribonucleotides (RNA)or deoxyribonucleotides (DNA), which can be single-stranded ordouble-stranded and which can contain non-natural or alterednucleotides. The terms “polynucleotide,” “nucleic acid sequence,” and“nucleotide sequence” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, either RNA or DNA. Theseterms refer to the primary structure of the molecule, and thus includedouble- and single-stranded DNA, and double- and single-stranded RNA.The terms include, as equivalents, analogs of either RNA or DNA madefrom nucleotide analogs and modified polynucleotides such as, though notlimited to methylated and/or capped polynucleotides. The polynucleotidecan be in any form, including but not limited to, plasmid, viral,chromosomal, EST, cDNA, mRNA, and rRNA.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The term“recombinant polypeptide” refers to a polypeptide that is produced byrecombinant techniques, wherein generally DNA or RNA encoding theexpressed protein is inserted into a suitable expression vector that isin turn used to transform a host cell to produce the polypeptide.Similarly, the terms “recombinant polynucleotide” or “recombinantnucleic acid” or “recombinant DNA” are produced by recombinanttechniques that are known to those of skill in the art.

The terms “homolog,” and “homologous” refer to a polynucleotide or apolypeptide comprising a sequence that is at least about 50 percent (%)identical to the corresponding polynucleotide or polypeptide sequence.Preferably homologous polynucleotides or polypeptides havepolynucleotide sequences or amino acid sequences that have at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or at least about 99% homology to thecorresponding amino acid sequence or polynucleotide sequence. As usedherein the terms sequence “homology” and sequence “identity” are usedinterchangeably. One of ordinary skill in the art is well aware ofmethods to determine homology between two or more sequences. Briefly,calculations of “homology” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Inone preferred embodiment, the length of a first sequence that is alignedfor comparison purposes is at least about 30%, preferably at least about40%, more preferably at least about 50%, even more preferably at leastabout 60%, and even more preferably at least about 70%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, or about 100% of the length of a second sequence. Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions of the first and second sequences are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps and the length of each gap, that need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent homology between two sequences can beaccomplished using a mathematical algorithm, such as BLAST (Altschul etal. (1990) J. Mol. Biol. 215(3):403-410). The percent homology betweentwo amino acid sequences also can be determined using the Needleman andWunsch algorithm that has been incorporated into the GAP program in theGCG software package, using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch (1970) J. Mol. Biol.48:444-453). The percent homology between two nucleotide sequences alsocan be determined using the GAP program in the GCG software package,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in theart can perform initial homology calculations and adjust the algorithmparameters accordingly. A preferred set of parameters (and the one thatshould be used if a practitioner is uncertain about which parametersshould be applied to determine if a molecule is within a homologylimitation of the claims) are a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frameshift gap penaltyof 5. Additional methods of sequence alignment are known in thebiotechnology arts (see, e.g., Rosenberg (2005) BMC Bioinformatics6:278; Altschul et al. (2005) FEBS J. 272(20):5101-5109).

The term “hybridizes under low stringency, medium stringency, highstringency, or very high stringency conditions” describes conditions forhybridization and washing. Guidance for performing hybridizationreactions can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methodsare described in that reference and either method can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions—6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions—6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions—6×SSC at about 45° C., followed by one or more washes in0.2.×SSC, 0.1% SDS at 65° C.; and (4) very high stringency hybridizationconditions—0.5M sodium phosphate, 7% SDS at 65° C., followed by one ormore washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions(4) are the preferred conditions unless otherwise specified.

An “endogenous” polypeptide refers to a polypeptide encoded by thegenome of the parental cell (or host cell). An “exogenous” polypeptiderefers to a polypeptide which is not encoded by the genome of theparental cell. A variant or mutant polypeptide is an example of anexogenous polypeptide. Thus, a non-naturally-occurring nucleic acidmolecule is considered to be exogenous to a cell once introduced intothe cell. A nucleic acid molecule that is naturally-occurring can alsobe exogenous to a particular cell. For example, an entire codingsequence isolated from cell X is an exogenous nucleic acid with respectto cell Y once that coding sequence is introduced into cell Y, even if Xand Y are the same cell type.

The term “overexpressed” means that a gene is caused to be transcribedat an elevated rate compared to the endogenous transcription rate forthat gene. In some examples, overexpression additionally includes anelevated rate of translation of the gene compared to the endogenoustranslation rate for that gene. Methods of testing for overexpressionare well known in the art, for example transcribed RNA levels can beassessed using rtPCR and protein levels can be assessed using SDS pagegel analysis.

The term “heterologous” means derived from a different organism,different cell type, or different species. As used herein it refers to anucleotide-, polynucleotide-, polypeptide- or protein sequence, notnaturally present in a given organism. For example, a polynucleotidesequence that is native to cyanobacteria can be introduced into a hostcell of E. coli by recombinant methods, and the polynucleotide fromcyanobacteria is then heterologous to the E. coli cell (e.g.,recombinant cell). The term “heterologous” may also be used withreference to a nucleotide-, polynucleotide-, polypeptide-, or proteinsequence which is present in a recombinant host cell in a non-nativestate. For example, a “heterologous” nucleotide, polynucleotide,polypeptide or protein sequence may be modified relative to the wildtype sequence naturally present in the corresponding wild type hostcell, e.g., a modification in the level of expression or in the sequenceof a nucleotide, polynucleotide, polypeptide or protein.

As used herein, the term “fragment” of a polypeptide refers to a shorterportion of a full-length polypeptide or protein ranging in size from twoamino acid residues to the entire amino acid sequence minus one aminoacid residue. In certain embodiments of the disclosure, a fragmentrefers to the entire amino acid sequence of a domain of a polypeptide orprotein (e.g., a substrate binding domain or a catalytic domain).

The term “mutagenesis” refers to a process by which the geneticinformation of an organism is changed in a stable manner. Mutagenesis ofa protein coding nucleic acid sequence produces a mutant protein.Mutagenesis also refers to changes in non-coding nucleic acid sequencesthat result in modified protein activity.

A “mutation”, as used herein, refers to a permanent change in a nucleicacid position of a gene or in an amino acid position of a polypeptide orprotein. Mutations include substitutions, additions, insertions, anddeletions. For example, a mutation in an amino acid position can be asubstitution of one type of amino acid with another type of amino acid(e.g., a serine (S) may be substituted with an alanine (A); a lysine (L)may be substituted with a threonine (T); etc.). As such, a polypeptideor a protein can have one or more mutations wherein one amino acid issubstituted with another amino acid.

As used herein, the term “gene” refers to nucleic acid sequencesencoding either an RNA product or a protein product, as well asoperably-linked nucleic acid sequences affecting the expression of theRNA or protein (e.g., such sequences include but are not limited topromoter or enhancer sequences) or operably-linked nucleic acidsequences encoding sequences that affect the expression of the RNA orprotein (e.g., such sequences include but are not limited to ribosomebinding sites or translational control sequences).

Expression control sequences are known in the art and include, forexample, promoters, enhancers, polyadenylation signals, transcriptionterminators, internal ribosome entry sites (IRES), and the like, thatprovide for the expression of the polynucleotide sequence in a hostcell. Expression control sequences interact specifically with cellularproteins involved in transcription (Maniatis et al. (1987) Science236:1237-1245). Exemplary expression control sequences are described in,for example, Goeddel, Gene Expression Technology: Methods in Enzymology,Vol. 185, Academic Press, San Diego, Calif. (1990). In the methods ofthe disclosure, an expression control sequence is operably linked to apolynucleotide sequence. By “operably linked” is meant that apolynucleotide sequence and an expression control sequence are connectedin such a way as to permit gene expression when the appropriatemolecules (e.g., transcriptional activator proteins) are bound to theexpression control sequence. Operably linked promoters are locatedupstream of the selected polynucleotide sequence in terms of thedirection of transcription and translation. Operably linked enhancerscan be located upstream, within, or downstream of the selectedpolynucleotide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid, i.e., a polynucleotidesequence, to which it has been linked. One type of useful vector is anepisome (i.e., a nucleic acid capable of extra-chromosomal replication).Useful vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked. Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors.” In general, expressionvectors of utility in recombinant DNA techniques are often in the formof “plasmids,” which refer generally to circular double stranded DNAloops that, in their vector form, are not bound to the chromosome. Otheruseful expression vectors are provided in linear form. Also included aresuch other forms of expression vectors that serve equivalent functionsand that have become known in the art subsequently hereto. In someembodiments, a recombinant vector further includes a promoter operablylinked to the polynucleotide sequence. In some embodiments, the promoteris a developmentally-regulated promoter, an organelle-specific promoter,a tissue-specific promoter, an inducible promoter, a constitutivepromoter, or a cell-specific promoter. The recombinant vector typicallycomprises at least one sequence selected from an expression controlsequence operatively coupled to the polynucleotide sequence; a selectionmarker operatively coupled to the polynucleotide sequence; a markersequence operatively coupled to the polynucleotide sequence; apurification moiety operatively coupled to the polynucleotide sequence;a secretion sequence operatively coupled to the polynucleotide sequence;and a targeting sequence operatively coupled to the polynucleotidesequence. In certain embodiments, the nucleotide sequence is stablyincorporated into the genomic DNA of the host cell, and the expressionof the nucleotide sequence is under the control of a regulated promoterregion. The expression vectors as used herein include a particularpolynucleotide sequence as described herein in a form suitable forexpression of the polynucleotide sequence in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,etc. The expression vectors described herein can be introduced into hostcells to produce polypeptides, including fusion polypeptides, encoded bythe polynucleotide sequences as described herein. Expression of genesencoding polypeptides in prokaryotes, for example, E. coli, is mostoften carried out with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionpolypeptides. Fusion vectors add a number of amino acids to apolypeptide encoded therein, usually to the amino- or carboxy-terminusof the recombinant polypeptide. Such fusion vectors typically serve oneor more of the following three purposes, including to increaseexpression of the recombinant polypeptide; to increase the solubility ofthe recombinant polypeptide; and to aid in the purification of therecombinant polypeptide by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantpolypeptide. This enables separation of the recombinant polypeptide fromthe fusion moiety after purification of the fusion polypeptide. Incertain embodiments, a polynucleotide sequence of the disclosure isoperably linked to a promoter derived from bacteriophage T5.

In certain embodiments, the host cell is a yeast cell, and theexpression vector is a yeast expression vector. Examples of vectors forexpression in yeast S. cerevisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234); pMFa (Kurjan et al. (1982) Cell 30:933-943);pJRY88 (Schultz et al. (1987) Gene 54: 113-123); pYES2 (InvitrogenCorp., San Diego, Calif.), and picZ (Invitrogen Corp., San Diego,Calif.). In other embodiments, the host cell is an insect cell, and theexpression vector is a baculovirus expression vector. Baculovirusvectors available for expression of proteins in cultured insect cells(e.g., Sf9 cells) include, for example, the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow et al.(1989) Virology 170:31-39). In yet another embodiment, thepolynucleotide sequences described herein can be expressed in mammaliancells using a mammalian expression vector. Other suitable expressionsystems for both prokaryotic and eukaryotic cells are well known in theart; see, e.g., Sambrook et al., “Molecular Cloning: A LaboratoryManual,” second edition, Cold Spring Harbor Laboratory, (1989).

As used herein, the term “CoA” or “acyl-CoA” refers to an acyl thioesterformed between the carbonyl carbon of alkyl chain and the sulfhydrylgroup of the 4′-phosphopantethionyl moiety of coenzyme A (CoA), whichhas the formula R—C(O)S-CoA, where R is any alkyl group having at least4 carbon atoms.

The term “ACP” means acyl carrier protein. ACP is a highly conservedcarrier of acyl intermediates during fatty acid biosynthesis, whereinthe growing chain is bound during synthesis as a thiol ester at thedistal thiol of a 4′-phosphopantetheine moiety. The protein exists intwo forms, i.e., apo-ACP (inactive in fatty acid biosynthesis) and ACPor holo-ACP (active in fatty acid biosynthesis). The terms “ACP” and“holo-ACP” are used interchangeably herein and refer to the active formof the protein. An enzyme called a phosphopantetheinyltransferase isinvolved in the conversion of the inactive apo-ACP to the activeholo-ACP. More specifically, ACP is expressed in the inactive apo-ACPform and a 4′-phosphopantetheine moiety must be post-translationallyattached to a conserved serine residue on the ACP by the action ofholo-acyl carrier protein synthase (ACPS), aphosphopantetheinyltransferase, in order to produce holo-ACP.

As used herein, the term “acyl-ACP” refers to an acyl thioester formedbetween the carbonyl carbon of an alkyl chain and the sulfhydryl groupof the phosphopantetheinyl moiety of an acyl carrier protein (ACP). Insome embodiments an ACP is an intermediate in the synthesis of fullysaturated acyl-ACPs. In other embodiments an ACP is an intermediate inthe synthesis of unsaturated acyl-ACPs. In some embodiments, the carbonchain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or 26 carbons.

As used herein, the term “fatty acid derivative” means a “fatty acid” ora “fatty acid derivative”, which may be referred to as a “fatty acid orderivative thereof”. The term “fatty acid” means a carboxylic acidhaving the formula RCOOH. R represents an aliphatic group, preferably analkyl group. R can include between about 4 and about 22 carbon atoms.Fatty acids can be saturated, monounsaturated, or polyunsaturated. A“fatty acid derivative” is a product made in part from the fatty acidbiosynthetic pathway of the production host organism (e.g., recombinanthost cell or microorganism). “Fatty acid derivatives” includes productsmade in part from ACP, acyl-ACP or acyl-ACP derivatives. Exemplary fattyacid derivatives include, for example, acyl-CoA, fatty acids, fattyaldehydes, short and long chain alcohols, fatty alcohols, hydrocarbons,esters (e.g., waxes, fatty acid esters, or fatty esters), terminalolefins, internal olefins, ketones as well as ω-OH fatty acids and ω-OHfatty acid derivatives thereof including α,ω-diacids, and otherbifunctional compounds.

As used herein, the term “fatty acid biosynthetic pathway” means abiosynthetic pathway that produces fatty acids and derivatives thereof.The fatty acid biosynthetic pathway may include additional enzymes toproduce fatty acids derivatives having desired characteristics.

The R group of a fatty acid can be a straight chain or a branched chain.Branched chains may have more than one point of branching and mayinclude cyclic branches. In some embodiments, the branched fatty acid isa C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀,C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, or a C₂₆ branched fatty acid. In otherembodiments, the branched fatty acid is a C₆, C₈, C₁₀, C₁₂, C₁₃, C₁₄,C₁₅, C₁₆, C₁₇, C₁₈, or C₂₀ branched fatty acid. In certain embodiments,the hydroxyl (OH) group of the branched fatty acid is in the omega (ω)position. In certain embodiments, the branched fatty acid is aniso-fatty acid or an anteiso-fatty acid. In exemplary embodiments, thebranched fatty acid is selected from iso-C_(7:0)-, iso-C_(8:0)-,iso-C_(9:0)-, iso-C_(10:0)-, iso-C_(12:0)-, iso-C_(13:0)-,iso-C_(15:0)-, iso-C_(17:0)-, iso-C_(18:0)-, iso-C_(19:0)-,iso-C_(20:0), anteiso-C_(7:0)-, anteiso-C_(9:0)-, anteiso-C_(11:0)-,anteiso-C_(13:0)-, anteiso-C_(15:0)-, anteiso-C_(17:0)-, andanteiso-C_(19:0) branched fatty acid.

The R group of a fatty acid can be saturated or unsaturated. Ifunsaturated, the R group can have one or more than one point ofunsaturation. In some embodiments, the unsaturated fatty acid is amonounsaturated fatty acid. In certain embodiments, the unsaturatedfatty acid is a C_(8:1)-, C_(9:1)-, C_(10:1)-, C_(11:1)-, C_(12:1)-,C_(13:1)-, C_(14:1)-, C_(15:1)-, C_(16:1)-, C_(17:1)-, C_(18:1)-,C_(19:1)-, C_(20:1)-, C_(21:1)-, C_(22:1)-, C_(23:1)-, C_(24:1)-,C_(25:1)-, or a C_(26:1) unsaturated fatty acid. In certain embodiments,the unsaturated fatty acid is C_(8:1), C_(10:1), C_(12:1), C_(14:1),C_(16:1), C_(18:1), or C_(20:1). In yet other embodiments, theunsaturated fatty acid is unsaturated at the omega-7 position. Incertain embodiments, the unsaturated fatty acid has a cis double bond.

As used herein, a “recombinant host cell” or “engineered host cell” is ahost cell, e.g., a microorganism that has been modified such that itproduces ω-hydroxylated fatty acids and ω-hydroxylated fatty acidderivatives including bi-functional fatty acid derivatives. In someembodiments, the recombinant host cell includes one or morepolynucleotides, each polynucleotide encoding a CYP153A-reductase hybridfusion polypeptide or variant thereof that has ω-hydroxylasebiosynthetic enzyme activity, wherein the recombinant host cell producesan ω-hydroxylated fatty acid and/or ω-hydroxylated fatty acid derivativeor composition thereof when cultured in the presence of a carbon sourceunder conditions effective to express the polynucleotides.

As used herein, the term “clone” typically refers to a cell or group ofcells descended from and essentially genetically identical to a singlecommon ancestor, for example, the bacteria of a cloned bacterial colonyarose from a single bacterial cell.

As used herein, the term “culture” typically refers to a liquid mediacomprising viable cells. In one embodiment, a culture comprises cellsreproducing in a predetermined culture media under controlledconditions, for example, a culture of recombinant host cells grown inliquid media comprising a selected carbon source and/or nitrogen.

The terms “culturing” or “cultivation” refers to growing a population ofcells (e.g., microbial cells) under suitable conditions in a liquid orsolid medium. In particular embodiments, culturing refers to thefermentative bioconversion of a substrate to an end-product. Culturingmedia are well known and individual components of such culture media areavailable from commercial sources, e.g., under the DIFCO media and BBLmedia. In one non-limiting example, the aqueous nutrient medium is a“rich medium” comprising complex sources of nitrogen, salts, and carbon,such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast extractof such a medium. In addition, the host cell can be engineered toassimilate carbon efficiently and use cellulosic materials as carbonsources according to methods described, for example, in U.S. Pat. Nos.5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030 and WO2010127318.In addition, the host cell can be engineered to express an invertase sothat sucrose can be used as a carbon source.

As used herein, the term “under conditions effective to express saidheterologous nucleotide sequences” means any conditions that allow ahost cell to produce a desired fatty acid derivative (e.g., ω-OH fattyacid and/or ω-OH fatty acid derivative). Suitable conditions include,for example, fermentation conditions.

As used herein, “modified” or an “altered level of” activity of aprotein, for example an enzyme, in a recombinant host cell refers to adifference in one or more characteristics in the activity determinedrelative to the parent or native host cell. Typically differences inactivity are determined between a recombinant host cell, having modifiedactivity, and the corresponding wild-type host cell (e.g., comparison ofa culture of a recombinant host cell relative to wild-type host cell).Modified activities can be the result of, for example, modified amountsof protein expressed by a recombinant host cell (e.g., as the result ofincreased or decreased number of copies of DNA sequences encoding theprotein, increased or decreased number of mRNA transcripts encoding theprotein, and/or increased or decreased amounts of protein translation ofthe protein from mRNA); changes in the structure of the protein (e.g.,changes to the primary structure, such as, changes to the protein'scoding sequence that result in changes in substrate specificity, changesin observed kinetic parameters); and changes in protein stability (e.g.,increased or decreased degradation of the protein). In some embodiments,the polypeptide is a mutant or a variant of any of the polypeptidesdescribed herein. In certain instances, the coding sequence for thepolypeptides as described herein are codon optimized for expression in aparticular host cell. For example, for expression in E. coli, one ormore codons can be optimized (Grosjean et al. (1982) Gene 18:199-209).

The term “regulatory sequences” as used herein typically refers to asequence of bases in DNA, operably-linked to DNA sequences encoding aprotein that ultimately controls the expression of the protein. Examplesof regulatory sequences include, but are not limited to, RNA promotersequences, transcription factor binding sequences, transcriptiontermination sequences, modulators of transcription (such as enhancerelements), nucleotide sequences that affect RNA stability, andtranslational regulatory sequences (such as, ribosome binding sites(e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences ineukaryotes), initiation codons, termination codons).

As used herein, the phrase “the expression of said nucleotide sequenceis modified relative to the wild type nucleotide sequence,” means anincrease or decrease in the level of expression and/or activity of anendogenous nucleotide sequence or the expression and/or activity of aheterologous or non-native polypeptide-encoding nucleotide sequence.

As used herein, the phrase “the activity of a CYP153A-reductase hybridfusion polypeptide sequence variant is modified relative to the activityof a CYP153A-reductase hybrid fusion polypeptide sequence (i.e., apolypeptide template) means an increase or decrease in the level ofactivity of an expressed polypeptide sequence variant in comparison toan expressed polypeptide sequence template. The polypeptide template isencoded by a nucleic acid template (i.e., a DNA template sequence). Anexample of a polypeptide sequence template is the hybrid cyp153A-RedRhFfusion protein sequence, wherein a cyp153A is fused with a reductasedomain. Another example of a polypeptide sequence template is SEQ ID NO:6. Another example of a polypeptide sequence template is SEQ ID NO: 38.Any polypeptide sequence can serve as a template including variants.

As used herein, the term “express” with respect to a polynucleotide isto cause it to function. A polynucleotide which encodes a polypeptide(or protein) will, when expressed, be transcribed and translated toproduce that polypeptide (or protein). As used herein, the term“overexpress” means to express (or cause to express) a polynucleotide orpolypeptide in a cell at a greater concentration than is normallyexpressed in a corresponding wild-type cell under the same conditions.In another embodiment, the term “overexpress” means to express (or causeto express) a polynucleotide or polypeptide in a cell at a greaterconcentration than it is normally expressed in a corresponding cell thatexpresses the template polynucleotide or template polypeptide sequenceunder the same conditions. An example of a template polypeptide sequenceis the CYP153A-RedRhF-hybrid fusion polypeptide.

The terms “altered level of expression” and “modified level ofexpression” are used interchangeably and mean that a polynucleotide,polypeptide, or fatty acid derivative is present in a differentconcentration in an engineered host cell as compared to itsconcentration in a corresponding wild-type cell under the sameconditions.

As used herein, the term “titer” refers to the quantity of ω-OH fattyacids and/or ω-OH fatty acid derivatives produced per unit volume ofhost cell culture. In any aspect of the compositions and methodsdescribed herein, an ω-OH fatty acid and/or ω-OH fatty acid derivativeis produced at a titer of about 25 mg/L, about 50 mg/L, about 75 mg/L,about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about200 mg/L, about 225 mg/L, about 250 mg/L, about 275 mg/L, about 300mg/L, about 325 mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L,about 425 mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L, about525 mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about 625mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L,about 750 mg/L, about 775 mg/L, about 800 mg/L, about 825 mg/L, about850 mg/L, about 875 mg/L, about 900 mg/L, about 925 mg/L, about 950mg/L, about 975 mg/L, about 1000 mg/L, about 1050 mg/L, about 1075 mg/L,about 1100 mg/L, about 1125 mg/L, about 1150 mg/L, about 1175 mg/L,about 1200 mg/L, about 1225 mg/L, about 1250 mg/L, about 1275 mg/L,about 1300 mg/L, about 1325 mg/L, about 1350 mg/L, about 1375 mg/L,about 1400 mg/L, about 1425 mg/L, about 1450 mg/L, about 1475 mg/L,about 1500 mg/L, about 1525 mg/L, about 1550 mg/L, about 1575 mg/L,about 1600 mg/L, about 1625 mg/L, about 1650 mg/L, about 1675 mg/L,about 1700 mg/L, about 1725 mg/L, about 1750 mg/L, about 1775 mg/L,about 1800 mg/L, about 1825 mg/L, about 1850 mg/L, about 1875 mg/L,about 1900 mg/L, about 1925 mg/L, about 1950 mg/L, about 1975 mg/L,about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L,50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or a range bounded byany two of the foregoing values. In other embodiments, an ω-OH fattyacid and/or ω-OH fatty acid derivative is produced at a titer of morethan 100 g/L, more than 200 g/L, more than 300 g/L, or higher, such as500 g/L, 700 g/L, 1000 g/L, 1200 g/L, 1500 g/L, or 2000 g/L. In oneembodiment, the titer of an ω-OH fatty acid and/or ω-OH fatty acidderivative produced by a recombinant host cell according to the methodsof the disclosure is from 5 g/L to 200 g/L, 10 g/L to 150 g/L, 20 g/L to120 g/L, 25 g/L to 110 g/L and 30 g/L to 100 g/L.

As used herein, the term “yield of the ω-OH fatty acids and/or ω-OHfatty acid derivatives produced by a host cell” refers to the efficiencyby which an input carbon source is converted to product (i.e., ω-OHfatty acids and/or ω-OH fatty acid derivatives) in a host cell. Hostcells engineered to produce ω-OH fatty acids and/or ω-OH fatty acidderivatives according to the methods of the disclosure have a yield ofat least 3%, at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, atleast 13%, at least 14%, at least 15%, at least 16%, at least 17%, atleast 18%, at least 19%, at least 20%, at least 21%, at least 22%, atleast 23%, at least 24%, at least 25%, at least 26%, at least 27%, atleast 28%, at least 29%, or at least 30% or a range bounded by any twoof the foregoing values. In other embodiments, an ω-OH fatty acid and/orω-OH fatty acid derivative is produced at a yield of more than 30%, 40%,50%, 60%, 70%, 80%, 90% or more. Alternatively, or in addition, theyield is about 30% or less, about 27% or less, about 25% or less, orabout 22% or less. Thus, the yield can be bounded by any two of theabove endpoints. For example, the yield of an ω-OH fatty acid and/orω-OH fatty acid derivative produced by the recombinant host cellaccording to the methods of the disclosure can be 5% to 15%, 10% to 25%,10% to 22%, 15% to 27%, 18% to 22%, 20% to 28%, 20% to 30%, 25% to 40%,or greater. An example of a preferred yield of an ω-OH fatty acid and/orω-OH fatty acid derivative produced by the recombinant host cellaccording to the methods of the disclosure is from 10% to 30%. Anotherexample of a preferred yield of an ω-OH fatty acid and/or ω-OH fattyacid derivative produced by the recombinant host cell according to themethods of the disclosure is from 10% to 40%. Another example of apreferred yield of an ω-OH fatty acid and/or ω-OH fatty acid derivativeproduced by the recombinant host cell according to the methods of thedisclosure is from 10% to 50%.

As used herein, the term “productivity” refers to the quantity of ω-OHfatty acids and/or ω-OH fatty acid derivatives produced per unit volumeof host cell culture per unit time. In any aspect of the compositionsand methods described herein, the productivity of an ω-OH fatty acidand/or ω-OH fatty acid derivative produced by a recombinant host cell isat least 100 mg/L/hour, at least 200 mg/L/hour, at least 300 mg/L/hour,at least 400 mg/L/hour, at least 500 mg/L/hour, at least 600 mg/L/hour,at least 700 mg/L/hour, at least 800 mg/L/hour, at least 900 mg/L/hour,at least 1000 mg/L/hour, at least 1100 mg/L/hour, at least 1200mg/L/hour, at least 1300 mg/L/hour, at least 1400 mg/L/hour, at least1500 mg/L/hour, at least 1600 mg/L/hour, at least 1700 mg/L/hour, atleast 1800 mg/L/hour, at least 1900 mg/L/hour, at least 2000 mg/L/hour,at least 2100 mg/L/hour, at least 2200 mg/L/hour, at least 2300mg/L/hour, at least 2400 mg/L/hour, or at least 2500 mg/L/hour. Inaddition, the productivity may be 2500 mg/L/hour or less, 2000mg/L/OD₆₀₀ or less, 1500 mg/L/OD₆₀₀ or less, 120 mg/L/hour, or less,1000 mg/L/hour or less, 800 mg/L/hour, or less, or 600 mg/L/hour orless. Thus, the productivity can be bounded by any two of the aboveendpoints. For example, the productivity can be 3 to 30 mg/L/hour, 6 to20 mg/L/hour, or 15 to 30 mg/L/hour. The preferred productivity of anω-OH fatty acid and/or ω-OH fatty acid derivative produced by arecombinant host cell according to the methods of the disclosure isselected from 500 mg/L/hour to 2500 mg/L/hour, or from 700 mg/L/hour to2000 mg/L/hour.

The terms “total fatty species (FAS)” and “total fatty acid product” maybe used interchangeably herein with reference to the total amount ofω-OH fatty acids and fatty acids present in a sample as evaluated byGC-FID as described in International Patent Application Publication WO2008/119082.

As used herein, the term “glucose utilization rate” means the amount ofglucose used by the culture per unit time, reported as grams/liter/hour(g/L/hr).

The term “carbon source from a renewable feedstock” when used alone orin reference to a feed source includes any biological material(including renewable feedstocks and/or biomass and/or waste products)from which carbon is derived except oleochemicals (i.e., refined oilsfrom plants and animals such as fatty acids, fatty acid esters, TAGs,hydroxy fatty acids, and the like) and petrochemicals (i.e., chemicalsderived from petroleum such as alkanes, alkenes, and the like). Thus,the term “carbon source from a renewable feedstock”, as used herein,excludes carbon derived from oleochemicals and petrochemicals. In someembodiments, the carbon source includes sugars or carbohydrates (e.g.,monosaccharides, disaccharides, or polysaccharides). In someembodiments, the carbon source is glucose and/or sucrose. In otherembodiments, the carbon source is derived from a renewable feedstocksuch as carbohydrates from corn, sugar cane, or lignocellulosic biomass;or waste products such as glycerol, flu-gas, syn-gas; or the reformationof organic materials such as biomass or natural gas; or is carbondioxide that is fixed photosynthetically. In other embodiments, abiomass is processed into a carbon source, which is suitable forbioconversion. In still other embodiments, the biomass does not requirefurther processing into a carbon source but can be used directly ascarbon source. An exemplary source of such biomass is plant matter orvegetation, such as switchgrass. Another exemplary carbon sourceincludes metabolic waste products, such as animal matter (e.g., cowmanure). Further exemplary sources of carbon include algae and othermarine plants. Another carbon source (including biomass) includes wasteproducts from industry, agriculture, forestry, and households,including, but not limited to, fermentation waste, fermentation biomass,glycerol/glycerine, ensilage, straw, lumber, sewage, garbage, maniplesolid waste, cellulosic urban waste, and food leftovers.

As used herein, the term “isolated,” with respect to products such asω-OH fatty acids and derivatives thereof refers to products that areseparated from cellular components, cell culture media, or chemical orsynthetic precursors. The fatty acids and derivatives thereof (e.g.,ω-OH fatty acid and/or ω-OH fatty acid derivatives) produced by themethods described herein can be relatively immiscible in thefermentation broth, as well as in the cytoplasm. Therefore, the fattyacids and derivatives thereof can collect in an organic phase eitherintracellularly or extracellularly.

As used herein, the terms “purify,” “purified,” or “purification” meanthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free (e.g., at least about 70% free, at least about75% free, at least about 85% free, at least about 90% free, at leastabout 95% free, at least about 97% free, at least about 99% free) fromother components with which they are associated. As used herein, theseterms also refer to the removal of contaminants from a sample. Forexample, the removal of contaminants can result in an increase in thepercentage of fatty acid derivatives such as ω-OH fatty acid and/or ω-OHfatty acid derivatives in a sample. For example, when a fatty acidderivative is produced in a recombinant host cell, the fatty acidderivative can be purified by the removal of host cell proteins or otherhost cell materials. After purification, the percentage of fatty acidderivative in the sample is increased. The terms “purify”, “purified,”and “purification” are relative terms which do not require absolutepurity. Thus, for example, when a fatty acid derivative is produced inrecombinant host cells, a purified fatty acid derivative is a fatty acidderivative that is substantially separated from other cellularcomponents (e.g., nucleic acids, polypeptides, lipids, carbohydrates, orother hydrocarbons).

Omega-Hydroxylated Fatty Acid and Fatty Acid Derivative Production

The disclosure provides for the production of ω-OH fatty acids and ω-OHfatty acid derivatives in a host cell. The ω-OH fatty acid productionmay be enhanced as a result of the expression of a CYP153A-reductasehybrid fusion polypeptide variant. The CYP153A-reductase hybrid fusionpolypeptide variants produce the ω-OH fatty acid derivatives andcompositions at a high titer. Herein, the CYP153A-reductase hybridfusion polypeptide variant is involved in a biosynthetic pathway for theproduction of ω-OH fatty acid derivatives; it may be used alone or incombination with other enzymes. For example, the CYP153A-reductasehybrid fusion polypeptide variant can be used in an engineeredbiosynthetic pathway wherein a thioesterase (i.e., naturally orheterologously expressed) enzyme converts an acyl-ACP to a fatty acid.The CYP153A-reductase hybrid fusion polypeptide variant can then convertthe fatty acid to an ω-OH fatty acid (see FIG. 1). Additional enzymes inthe pathway can convert ω-OH fatty acids into other bi-functional fattyacid derivatives such as α,ω-diacids.

More specifically, a CYP153A-reductase hybrid fusion polypeptide is apolypeptide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO: 6 and serves as a template sequence to introducemutations in order to create variants with improved enzymatic activityfor the production of ω-OH fatty acids and fatty acid derivatives. TheCYP153A-reductase hybrid fusion polypeptide is self-sufficient andpossesses ω-hydroxylase enzymatic activity that catalyzes the reactionof a fatty acid to an ω-OH fatty acid. An example of a CYP153A-reductasehybrid fusion polypeptide is a hybrid cyp153A-RedRhF-type fusionpolypeptide. In one embodiment, term CYP153A-reductase hybrid fusionpolypeptide variant refers to a modified CYP153A-reductase hybrid fusionpolypeptide that has at least one mutation in its amino acid sequenceincluding, but not limited to, a mutation at amino acid position 796,141, 231, 27, 82, 178, 309, 407, 415, 516 and 666 or a combinationthereof. The expression of the CYP153A-reductase hybrid fusionpolypeptide variant in recombinant host cells results in improved titer,yield and/or productivity of ω-OH fatty acids and/or ω-OH fatty acidderivatives or compositions thereof when compared to the expression ofthe CYP153A-reductase hybrid fusion polypeptide in a corresponding hostcell.

An example of a CYP153A-reductase hybrid fusion polypeptide variant isSEQ ID NO: 38, which has a mutation in position 796, wherein an alanineis replaced with a valine. This CYP153A-reductase hybrid fusionpolypeptide variant has a polypeptide sequence that has at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to SEQ ID NO: 38 and it further serves atemplate sequence to create additional mutations or additional variants.Similarly, this CYP153A-reductase hybrid fusion polypeptide variant isself-sufficient and possesses ω-hydroxylase enzymatic activity thatcatalyzes the reaction of a fatty acid to an ω-OH fatty acid. In oneembodiment, term CYP153A-reductase hybrid fusion polypeptide variantrefers to a modified CYP153A-reductase hybrid fusion polypeptide thathas at least one additional mutation in its amino acid sequenceincluding, but not limited to, a mutation at amino acid position 747,12, 327, 14, 61, 28, 13, 771, 119, 10, 11, 28, 745, 9, 770, 413, 784,749, 231, 233, 757 and 703 or a combination thereof. The expression ofthe CYP153A-reductase hybrid fusion polypeptide variant in recombinanthost cells results in improved titer, yield and/or productivity of ω-OHfatty acids and/or ω-OH fatty acid derivatives when compared to theexpression of the CYP153A-reductase hybrid fusion polypeptide (SEQ IDNo: 6) in a corresponding host cell.

When a cell has been transformed with a CYP153A-reductase hybrid fusionpolypeptide variant it is a cell that expresses the CYP153A-reductasehybrid fusion polypeptide variant (e.g., a recombinant cell). In oneembodiment, the titer and/or yield of an ω-OH fatty acid produced by acell that expresses the CYP153A-reductase hybrid fusion polypeptidevariant is at least twice that of a corresponding cell that expressesthe CYP153A-reductase hybrid fusion polypeptide. In a host such asEscherichia coli, ω-OH fatty acids may be converted to bi-functionalfatty acid derivatives by naturally or heterologously expressed enzymes.In another embodiment, the titer and/or yield of an ω-OH fatty acid orderivative thereof produced by a cell that expresses theCYP153A-reductase hybrid fusion polypeptide variant is at least about 1times, at least about 2 times, at least about 3 times, at least about 4times, at least about 5 times, at least about 6 times, at least about 7times, at least about 8 times, at least about 9 times, or at least about10 times greater than that of a corresponding cell that expresses theCYP153A-reductase hybrid fusion polypeptide. In one embodiment, thetiter and/or yield of an ω-OH fatty acid or derivative thereof producedby a cell expressing the CYP153A-reductase hybrid fusion polypeptidevariant is at least about 1 percent, at least about 2 percent, at leastabout 3 percent, at least about 4 percent, at least about 5 percent, atleast about 6 percent, at least about 7 percent, at least about 8percent, at least about 9 percent, or at least about 10 percent greaterthan that of a corresponding cell that expresses the CYP153A-reductasehybrid fusion polypeptide. In another embodiment, the titer and/or yieldof an ω-OH fatty acid or derivative thereof produced in a recombinantcell due to the expression of a CYP153A-reductase hybrid fusionpolypeptide variant is at least about 20 percent to at least about 80percent greater than that of a corresponding cell that expresses theCYP153A-reductase hybrid fusion polypeptide. In some embodiments, thetiter and/or yield of an ω-OH fatty acid produced by a cell is at leastabout 20 percent, at least about 25 percent, at least about 30 percent,at least about 35 percent, at least about 40 percent, at least about 45percent, at least about 50 percent, at least about 55 percent, at leastabout 60 percent, at least about 65 percent, at least about 70 percent,at least about 75 percent, at least about 80 percent, at least about 85percent, at least about 90 percent, at least about 95 percent, at leastabout 97 percent, at least about 98 percent, or at least about 100percent greater than that of the corresponding cell that expresses theCYP153A-reductase hybrid fusion polypeptide.

Thus, the disclosure provides recombinant host cells, which have beenengineered to express a CYP153A-reductase hybrid fusion polypeptidevariant to produce ω-OH fatty acids or derivatives thereof. Thebiosynthesis of ω-OH fatty acids is enhanced relative to theCYP153A-reductase hybrid fusion polypeptide-expressing host cells, i.e.,host cells that express the CYP153A-reductase hybrid fusion polypeptidebased on SEQ ID NO: 6 (i.e., template polypeptide) or other polypeptideswith the same enzymatic function. A variety of different host cells canbe modified to express a CYP153A-reductase hybrid fusion polypeptidevariant such as those described herein, resulting in recombinant hostcells suitable for the enhanced production of ω-OH fatty acid and ω-OHfatty acid derivatives or compositions thereof. Examples of ω-OH fattyacids that are produced are C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄,C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1),C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1),C_(19:1) and/or C_(20:1) fatty acids. In one embodiment, such ω-OH fattyacids are ω-OH C_(8:0) fatty acids, ω-OH C_(10:0) fatty acids, ω-OHC_(12:0) fatty acids, ω-OH C_(14:0) fatty acids, ω-OH C_(16:0) fattyacids, ω-OH C_(18:0) fatty acids, ω-OH C_(20:0) fatty acids, ω-OHC_(8:1) fatty acids, ω-OH C_(10:1) fatty acids, ω-OH C_(12:1) fattyacids, ω-OH C_(14:1) fatty acids, ω-OH C_(16:1) fatty acids, ω-OHC_(18:1) fatty acids, ω-OH C_(20:1) fatty acids, and the like. It isunderstood that a variety of cells can provide sources of geneticmaterial, including polynucleotide sequences that encode polypeptidessuitable for use in a recombinant host cell as described herein.

Pathway Engineering and Enzymatic Activities

Fatty acid synthesis is one of the most conserved systems of thebacterial biosynthetic machinery. The fatty acid synthase (FAS)multi-enzyme complex is present in all bacteria and eukaryotes. Most ofthe FAS related genes are indispensable for cell growth and survival.Eukaryotic and bacterial FAS drive essentially the same type ofbiochemical transformation. In eukaryotes, FAS is referred to as FAS Iand most of its catalytic domains are encoded by one polypeptide chain(non-dissociable). In prokaryotes such as bacteria, FAS is referred toas FASII and its individual enzymes and carrier proteins are encoded byseparate genes coding for discrete (dissociable) proteins. As such,FASII is a complex system with significant variations and distinctpeculiarities.

The acyl carrier protein (ACP) along with the enzymes in a FAS pathwaycontrol the length, degree of saturation and branching of the fattyacids produced in a native organism. The steps in this pathway arecatalyzed by enzymes of the fatty acid biosynthesis (FAB) and acetyl-CoAcarboxylase (ACC) gene families. For example, enzymes that can beincluded in a FAS pathway include AccABCD, FabD, FabH, FabG, FabA, FabZ,FabI, FabK, FabL, FabM, FabB, and FabF. Depending upon the desiredproduct one or more of these genes can be attenuated or over-expressed.As such, prokaryotes have been engineered to increase production offatty acid derivatives from renewable feedstock such as glucose or othercarbon sources. Herein the major goal is to increase the activity of keycontrol enzymes that regulate the production of fatty acid derivativesin order to convert the bacterial strain into a microbial factory forfatty acid derivative production, including fatty acid methyl esters(FAMEs), fatty acid ethyl esters (FAEEs), and fatty alcohols (FALC)(see, e.g., U.S. Pat. No. 8,283,143, incorporated by reference herein).

The present disclosure identifies CYP153A-reductase hybrid fusionpolynucleotides that encode polypeptides of enzymatic function in orderto modify enzymatic pathways for the production of desirable compoundssuch as ω-OH fatty acids and ω-OH fatty acid derivatives. Thesepolypeptides, which are identified herein by Enzyme Accession Numbers(EC Numbers), are useful for engineering fatty acid pathways that leadto production of ω-OH fatty acids and other bi-functional molecules suchas ω-OH fatty acid derivatives like α,ω-diacids (see FIG. 1).

In one embodiment, pathways are depicted in FIG. 1 that use a carbonsource derived from a renewable feedstock such as glucose to produceω-OH fatty acid derivatives. A carbohydrate (e.g., glucose) is convertedto an acyl-thioester such as an acyl-ACP by the native organism (seestep 1 in FIG. 1). Polynucleotides that code for polypeptides with fattyacid degradation enzyme activity can be optionally attenuated dependingon the desired product (see Examples, infra). Non-limiting examples ofsuch polypeptides are acyl-CoA synthetase (FadD) and acyl-CoAdehydrogenase (FadE). Table 1 provides a comprehensive list of enzymaticactivity (infra) within the metabolic pathway, including various fattyacid degradation enzymes that can be optionally attenuated according tomethods known in the art (see, e.g., U.S. Pat. No. 8,283,143, supra).

For example, FadR (see Table 1, infra) is a key regulatory factorinvolved in fatty acid degradation and fatty acid biosynthetic pathways(Cronan et al., Mol. Microbiol., 29(4): 937-943 (1998)). The E. colienzyme FadD (see Table 1, infra) and the fatty acid transport proteinFadL are components of a fatty acid uptake system. FadL mediatestransport of fatty acids into the bacterial cell, and FadD mediatesformation of acyl-CoA esters. When no other carbon source is available,exogenous fatty acids are taken up by bacteria and converted to acyl-CoAesters, which can bind to the transcription factor FadR and depress theexpression of the fad genes that encode proteins responsible for fattyacid transport (FadL), activation (FadD), and β-oxidation (FadA, FadB,and FadE). When alternative sources of carbon are available, bacteriasynthesize fatty acids as acyl-ACPs, which are used for phospholipidsynthesis, but are not substrates for β-oxidation. Thus, acyl-CoA andacyl-ACP are both independent sources of fatty acids that can result indifferent end-products (Caviglia et al., J. Biol. Chem., 279(12):1163-1169 (2004)).

TABLE 1 Enzymatic Activities Gene Source Designation Organism EnzymeName Accession # EC Number Exemplary Use Fatty Acid Production IncreaseaccA E. coli, Acetyl-CoA AAC73296, 6.4.1.2 increase Malonyl-CoALactococci carboxylase, NP_414727 production subunit A(carboxyltransferase alpha) accB E. coli, Acetyl-CoA NP_417721 6.4.1.2increase Malonyl-CoA Lactococci carboxylase, subunit production B (BCCP:biotin carboxyl carrier protein) accC E. coli, Acetyl-CoA NP_4177226.4.1.2, increase Malonyl-CoA Lactococci carboxylase, subunit 6.3.4.14production C (biotin carboxylase) accD E. coli, Acetyl-CoA NP_4168196.4.1.2 increase Malonyl-CoA Lactococci carboxylase, production subunitD (carboxyltransferase beta) fadD E. coli W3110 acyl-CoA synthaseAP_002424 2.3.1.86, increase Fatty acid 6.2.1.3 production fabA E. coliK12 β-hydroxydecanoyl NP_415474 4.2.1.60 increase fatty acyl- thioesterACP/CoA production dehydratase/isomerase fabB E. coli 3-oxoacyl-[acyl-BAA16180 2.3.1.41 increase fatty acyl- carrier-protein] ACP/CoAproduction synthase I fabD E. coli K12 [acyl-carrier-protein] AAC741762.3.1.39 increase fatty acyl- S-malonyltransferase ACP/CoA productionfabF E. coli K12 3-oxoacyl-[acyl- AAC74179 2.3.1.179 increase fattyacyl- carrier-protein] ACP/CoA production synthase II fabG E. coli K123-oxoacyl-[acyl- AAC74177 1.1.1.100 increase fatty acyl- carrierprotein] ACP/CoA production reductase fabH E. coli K12 3-oxoacyl-[acyl-AAC74175 2.3.1.180 increase fatty acyl- carrier-protein] ACP/CoAproduction synthase III fabI E. coli K12 enoyl-[acyl-carrier- NP_4158041.3.1.9 increase fatty acyl- protein] reductase ACP/CoA production fabRE. coli K12 Transcriptional NP_418398 none modulate unsaturatedRepressor fatty acid production fabV Vibrio choleraeenoyl-[acyl-carrier- YP_001217283 1.3.1.9 increase fatty acyl- protein]reductase ACP/CoA production fabZ E. coli K12 (3R)-hydroxymyristolNP_414722 4.2.1.— increase fatty acyl- acyl carrier protein ACP/CoAproduction dehydratase fadE E. coli K13 acyl-CoA AAC73325 1.3.99.3,reduce fatty acid dehydrogenase 1.3.99.— degradation fadD E. coli K12acyl-CoA synthetase NP_416319 6.2.1.3 reduce fatty acid degradation fadAE. coli K12 3-ketoacyl-CoA YP_02627 2.3.1.16 reduce fatty acid thiolasedegradation fadB E. coli K12 enoyl-CoA hydratase, NP_418288 4.2.1.17,reduce fatty acid 3-OH acyl-CoA 5.1.2.3, degradation epimerase/ 1.1.1.35dehydrogenase fadR E. coli transcriptional NP_415705 none Block orreverse regulatory protein fatty acid degradation Chain Length ControltesA (with or E. coli thioesterase - leader P0ADA1 3.1.2.—, C18 ChainLength without leader sequence is amino 3.1.1.5 sequence) acids 1-26tesA (without E. coli thioesterase AAC73596, 3.1.2.—, C18:1 Chain Lengthleader NP_415027 3.1.1.5 sequence) tesA (mutant of E. coli thioesteraseL109P 3.1.2.—, <C18 Chain Length E. coli 3.1.1.5 thioesterase Icomplexed with octanoic acid) fatB1 Umbellularia californicathioesterase Q41635 3.1.2.14 C12:0 Chain Length fatB2 Cuphea hookerianathioesterase AAC49269 3.1.2.14 C8:0-C10:0 Chain Length fatB3 Cupheahookeriana thioesterase AAC72881 3.1.2.14 C14:0-C16:0 Chain Length fatBCinnamomum camphora thioesterase Q39473 3.1.2.14 C14:0 Chain Length fatBArabidopsis thaliana thioesterase CAA85388 3.1.2.14 C16:l Chain LengthfatB1 Umbellularia californica thioesterase Q41635 3.1.2.14 C12:0 ChainLength fatA1 Helianthus annuus thioesterase AAL79361 3.1.2.14 C18:1Chain Length fatA Arabidopsis thaliana thioesterase NP_189147, 3.1.2.14C18:1 Chain Length NP_193041 fatA Brassica juncea thioesterase CAC391063.1.2.14 C18:1 Chain Length fatA Cuphea hookeriana thioesterase AAC728833.1.2.14 C18:1 Chain Length tes Photobacterium profundum thioesteraseYP_130990 3.1.2.14 Chain Length tesB E. coli thioesterase NP_4149863.1.2.14 Chain Length fadM E. coli thioesterase NP_414977 3.1.2.14 ChainLength yciA E. coli thioesterase NP_415769 3.1.2.14 Chain Length ybgC E.coli thioesterase NP_415264 3.1.2.14 Chain Length Saturation LevelControl Sfa E. coli Suppressor of fabA AAN79592, none increase AAC44390monounsaturated fatty acids fabA E. coli K12 β-hydroxydecanoyl NP_4154744.2.1.60 produce unsaturated thioester fatty acids dehydratase/isomeraseGnsA E. coli suppressors of the ABD18647.1 none increase unsaturatedsecG null mutation fatty acid esters GnsB E. coli suppressors of theAAC74076.1 none increase unsaturated secG null mutation fatty acidesters fabB E. coli 3-oxoacyl-[acyl- BAA16180 2.3.1.41 modulateunsaturated carrier-protein] fatty acid production synthase I desBacillus subtilis D5 fatty acyl O34653 1.14.19 modulate unsaturateddesaturase fatty acid production Ester Production AT3G51970 Arabidopsisthaliana long-chain-alcohol O- NP_190765 2.3.1.26 ester productionfatty-acyltransferase ELO1 Pichia angusta Fatty acid elongase BAD982512.3.1.— produce very long chain length fatty acids plsC Saccharomycescerevisiae acyltransferase AAA16514 2.3.1.51 ester production DAGAT/DGATArabidopsis thaliana diacylglycerol AAF19262 2.3.1.20 ester productionacyltransferase hWS Homo sapiens acyl-CoA wax AAX48018 2.3.1.20 esterproduction alcohol acyltransferase aft1 Acinetobacter sp. bifunctionalwax AAO17391 2.3.1.20 ester production ADP1 ester synthase/acyl- CoA:diacylglycerol acyltransferase ES9 Marinobacter hydrocarbonoclasticuswax ester synthase ABO21021 2.3.1.20 ester production mWS Simmondsiachinensis wax ester synthase AAD38041 2.3.1.— ester production FattyAlcohol Output thioesterases increase fatty (see above) acid/fattyalcohol production BmFAR Bombyxmori FAR (fatty alcohol BAC79425 1.1.1.—convert acyl-CoA forming acyl-CoA to fatty alcohol reductase) acr1Acinetobacter sp. acyl-CoA reductase YP_047869 1.2.1.42 reduce fattyacyl-CoA to ADP1 fatty aldehydes yqhD E. coli W3110 alcohol AP_0035621.1.—.— reduce fatty aldehydes to dehydrogenase fatty alcohols; increasefatty alcohol production alrA Acinetobacter sp. alcohol CAG70252 1.1.—.—reduce fatty aldehydes to ADP1 dehydrogenase fatty alcohols BmFARBombyxmori FAR (fatty alcohol BAC79425 1.1.1.— reduce fatty acyl-CoA toforming acyl-CoA reductase) fatty alcohol GTNG_1865 Geobacillusthermodenitrificans Long-chain aldehyde YP_001125970 1.2.1.3 reducefatty aldehydes to NG80-2 dehydrogenase fatty alcohols AAR Synechococcuselongatus Acyl-ACP reductase YP_400611 1.2.1.42 reduce fatty acyl-ACP/CoA to fatty aldehydes carB Mycobacterium smegmatis carboxylic acidYP_889972 6.2.1.3, reduce fatty acids reductase protein 1.2.1.42 tofatty aldehyde FadD E. coli K12 acyl-CoA synthetase NP_416319 6.2.1.3activates fatty acids to fatty acyl-CoAs atoB Erwinia carotovoraacetyl-CoA YP_049388 2.3.1.9 production of butanol acetyltransferase hbdButyrivibrio fibrisolvens Beta-hydroxybutyryl- BAD51424 1.1.1.157production of butanol CoA dehydrogenase CPE0095 Clostridium perfringenscrotonasebutyryl- BAB79801 4.2.1.55 production of butanol CoAdehydryogenase bcd Clostridium beijerinckii butyryl-CoA AAM145831.3.99.2 production of butanol dehydryogenase ALDH Clostridiumbeijerinckii coenzyme A- AAT66436 1.2.1.3 production of butanolacylating aldehyde dehydrogenase AdhE E. coli CFT073 aldehyde-alcoholAAN80172 1.1.1.1 production of butanol dehydrogenase 1.2.1.10 FattyAlcohol Acetyl Ester Output thioesterases modify output (see above) acr1Acinetobacter sp. acyl-CoA reductase YP_047869 1.2.1.42 modify outputADP1 yqhD E. Coli K12 alcohol AP_003562 1.1.—.— modify outputdehydrogenase AAT Fragaria × alcohol O- AAG13130 2.3.1.84 modify outputananassa acetyltransferase Terminal Olefin Output OleT Jeotgalicoccussp. Fatty acid HQ709266 1.11.2.4 decarboxylate fatty decarboxylase acidsProduct Export AtMRP5 Arabidopsis thaliana Arabidopsis thalianaNP_171908 none modify product export multidrug resistance- amountassociated AmiS2 Rhodococcus sp. ABC transporter JC5491 none modifyproduct export AmiS2 amount AtPGP1 Arabidopsis thaliana Arabidopsisthaliana NP_181228 none modify product export p glycoprotein 1 amountAcrA Candidatus Protochlamydiaamoebophila putative multidrug- CAF23274none modify product export UWE25 efflux transport amount protein acrAAcrB Candidatus Protochlamydiaamoebophila probable multidrug- CAF23275none modify product export UWE25 efflux transport amount protein, acrBTolC Francisella tularensis subsp. Outer membrane ABD59001 none modifyproduct export novicida protein [Cell amount envelope biogenesis, AcrEShigella sonnei transmembrane YP_312213 none modify product export Ss046protein affects amount septum formation and cell membrane permeabilityAcrF E. coli Acriflavine resistance P24181 none modify product exportprotein F amount tll1619 Thermosynechococcus elongatus multidrug effluxNP_682409.1 none modify product export [BP-1] transporter amount tll0139Thermosynechococcus elongatus multidrug efflux NP_680930.1 none modifyproduct export [BP-1] transporter amount Fermentation replicationincrease output checkpoint efficiency genes umuD Shigella sonnei DNApolymerase V, YP_310132 3.4.21.— increase output Ss046 subunitefficiency umuC E. coli DNA polymerase V, ABC42261 2.7.7.7 increaseoutput subunit efficiency pntA, pntB Shigella flexneri NADH:NADPHP07001, 1.6.1.2 increase output transhydrogenase P0AB70 efficiency(alpha and beta subunits) Other fabK Streptococcus pneumoniaetrans-2-enoyl-ACP AAF98273 1.3.1.9 Contributes to reductase II fattyacid biosynthesis fabL Bacillus licheniformis enoyl-(acyl carrierAAU39821 1.3.1.9 Contributes to DSM 13 protein) reductase fatty acidbiosynthesis fabM Streptococcus mutans trans-2, cis-3- DAA05501 4.2.1.17Contributes to decenoyl-ACP fatty acid biosynthesis isomerase

FIG. 1 shows an exemplary pathway where an acyl thioester such as anacyl-ACP can be converted to a C₁₂ or C_(16:1) fatty acid (FFA) as aprecursor intermediate. In step 1 of FIG. 1, a thioesterase is employedto covert an acyl-ACP to a FFA. In certain embodiments, the geneencoding a thioesterase is tesA, ‘tesA, tesB, fatB1, fatB2, fatB3,fatA1, or fatA (see also Table 1 that shows polypeptides that have theenzymatic activity of a thioesterase that can be used to catalyze thisstep, supra). In step 2, a CYP153A-reductase hybrid fusion polypeptideor variant thereof is used to generate ω-OH fatty acids (ω-OH FFAs) fromfatty acids. Other bifunctional molecules can be produced downstream inthe pathway, for example α,ω-diacids or other ω-OH fatty acidderivatives, depending on the enzymatic functionalities that are presentin the pathway.

CYP153A-Reductase Hybrid Fusion Polypeptides

ω-Hydroxylases (or ω-oxygenases) include certain non-heme di-ironoxygenases (e.g., alkB from Pseudomonas putida GPol) and certainheme-type P450 oxygenases (e.g., ω-hydroxylases such as cyp153A fromMarinobacter aquaeolei). P450s are ubiquitously distributed enzymes,which possess high complexity and display a broad field of activity.They are proteins encoded by a superfamily of genes that convert a broadvariety of substrates and catalyze a variety of chemical reactions.Cyp153A is a sub-family of soluble bacterial cytochrome P450s thathydroxylate hydrocarbon chains with high selectivity for the ω-position(van Beilen et al. (2006) Appl. Environ. Microbiol. 72:59-65). Membersof the cyp153A family have been shown in vitro to selectivelyhydroxylate the ω-position of alkanes, fatty acids or fatty alcohols,for example cyp153A6 from Mycobacterium sp. HXN-1500 (Funhoff et al.(2006) J Bacteriol. 188:5220-5227), cyp153A16 from Mycobacterium marinumand cyp153A from Polaromonas sp. JS666 (Scheps et al. (2011) Org.Biomol. Chem. 9:6727-6733) as well as cyp153A from Marinobacter aquaeoli(Honda-Malca et al. (2012) Chem. Commun. 48:5115-5117). Tables 2A and 2Bbelow show examples of enzymes and redox partners that haveω-hydroxylase enzymatic activity that can be used to produce ω-OH fattyacids and ω-OH fatty acid derivatives.

TABLE 2A Examples of ω-Hydroxylase Enzymatic Activity (P450) (EC1.14.15.3) Gene Hydroxylation Designation Source Organism Accession No.Redox System Position cyp153A Acinetobacter sp. BAE78452 operon withferredoxin and ω-hydroxylase (aciA) OC4 ferredoxin reductase cyp153A16Mycobacterium marinum M YP_001851443 operon with ferredoxin andω-hydroxylase ferredoxin reductase cyp153A6 Mycobacterium AJ833989operon with ferredoxin and ω-hydroxylase sp. HXN-1500 ferredoxinreductase cyp153A Marinobacter aquaeolei YP_957888 operon withferredoxin and ω -hydroxylase VT8 ferredoxin reductase alkB Pseudomonasputida CAB54050 requires rubredoxin and ω-hydroxylase GPo1 rubredoxinreductase alkB Pseudomonas fluorescens CAB51045 requires rubredoxin andω-hydroxylase CHA0 rubredoxin reductase alkM Acinetobacter baylyiYP_046098 requires rubredoxin and ω-hydroxylase rubredoxin reductasealkB Gordonia sp. ADT82701 requires rubredoxin and ω-hydroxylase SoGcrubredoxin reductase alkW1 Dietzia sp. HQ850582 c-terminal rubredoxinω-hydroxylase DQ12-45-1b fusion, requires rubredoxin reductase alkBPseudomonas putida CAB54050 requires rubredoxin and ω-hydroxylase GPo1rubredoxin reductase alkB Pseudomonas fluorescens CAB51045 requiresrubredoxin and ω-hydroxylase CHA0 rubredoxin reductase

TABLE 2B Examples of Redox Partners for ω-Hydroxylase Enzymatic Activity(P450) (EC 1.14.15.3) Designation/Name Organism Accession # ferredoxin,ferredoxin Acinetobacter sp. OC4 BAE78451, BAE78453 reductaseferredoxin, ferredoxin Mycobacterium marinum M YP_001851444,YP_001851442 reductase ferredoxin, ferredoxin Marinobacter aquaeoli VT8YP_957887, YP_957889 reductase alkG, alkT Pseudomonas putida GPo1CAB54052, CAB54063 rubA, rubB Acinetobacter baylyi ADP1 CAA86925,CAA86926

As with all cytochrome P450s, Cyp153A ω-hydroxylases require electronsfor their catalytic activity, which are provided via specific redoxproteins such as ferredoxin and ferredoxin reductase. These are discreteproteins interacting with cyp153A. A self-sufficient hybrid (chimeric)cyp153A oxygenase (i.e., an oxygenase that does not require discreteferredoxin and ferredoxin reductase proteins for activity) haspreviously been created by fusing cyp153A from Alcanivorax borkumensisSK2 (Kubota et al. (2005) Biosci. Biotechnol. Biochem. 69:2421-2430;Fujita et al. (2009) Biosci. Biotechnol. Biochem. 73:1825-1830) with thereductase domain from P450RhF, which includes flavin mononucleotide(FMN) and NADPH-binding sites and a [2FeS] ferredoxin center (Hunter etal. (2005) FEBS Lett. 579:2215-2220). P450RhF belongs to the class-IP450-fused PFOR (DeMot and Parret (2003) Trends Microbiol. 10: 502).This hybrid cyp153A-RedRhF fusion protein was shown in in vitrobiotransformations to hydroxylate octane in the ω-position and alsohydroxylate other compounds such as cyclohexane or butylbenzene. Otherself-sufficient hybrid (chimeric) cyp153A oxygenases have been createdby fusing cyp153A from Marinobacter aquaeolei with the reductase domainsfrom P450RhF and P450-BM3 (Scheps et al. (2013) Microb. Biotechnol.6:694-707). Examples of natural P450-Reductase fusion proteins are shownin Tables 2C and 2D below.

TABLE 2C Examples of Self-Sufficient ω-1, ω-2, ω- 3-Hydroxylase (EC1.14.14.1) Fusion Proteins Gene Hydroxylation Designation SourceOrganism Accession No. Redox System Position P450-BM3 Bacillusmegaterium AAA87602 fusion protein with ω-1, -2, -3 (cyp102A1) reductasedomain hydroxylation yrhJ Bacillus subtilis NP_390594 fusion proteinwith ω-1, -2, -3 (cyp102A3) reductase domain hydroxylation yrhJ Bacilluslicheniformis AAU41718 fusion protein with ω-1, -2, -3 (cyp102A7)reductase domain hydroxylation

TABLE 2D Examples of Self-Sufficient Class-I P450-Fused PFOR FusionProteins Designation/Name Organism Accession # P450RhF Rhodococcus sp.AAM67416 NCIMB 9784 REQ_44300 Rhodococcus equi YP_004009071 103SHMPREF0018_01193 Acinetobacter radioresistens ZP_06072406 SH164 BMAA1669Burkholderia mallei YP_106239 ATCC 23344 Rmet_4932 Cupriavidusmetallidurans YP_587063 CH34 H16_B1279 Ralstonia eutropha YP_840799 H16

Given their high selectivity towards the ω-position of hydrocarbonchains, the cyp153A family oxygenases appeared to be good examples ofsuitable candidates to produce α,ω-bifunctional fatty acid derivativesfrom a renewable carbon source. This would allow for the development ofcommercially feasible processes to produce these valuable compounds.Yet, as with other cytochrome P450s, the cyp153A family proteins have sofar mostly been applied to in vitro experiments with purified enzymes orcrude cell lysates or in resting cell biotransformations to which fattyacid derivatives or hydrocarbons are added exogenously (Kubota et al.,Fujita et al., Honda-Malca et al., Scheps et al., supra). However, thehybrid fusion-employing in vitro procedures or resting cellbiotransformations are not conducive to large scale and cost-efficientproduction of ω-hydroxy fatty acid derivatives. The widely acceptedknowledge in the art is that many cytochrome P450s as well as alkB-typeω-hydroxylases are not easy to express functionally in recombinantmicroorganisms because the enzymes are often inactive and theirchemistry has been difficult to elucidate. In fact, the only in vivowork using a renewable carbon source other than fatty acid-derivativesthat has so far been attempted employed alkB ω-hydroxylase and achievedonly low titer of ω-hydroxy fatty acid derivatives in a high celldensity fermentation (WO2013/024114A2).

The present disclosure provides CYP153A-reductase hybrid fusion proteinvariants that are capable of efficiently producing ω-hydroxy fatty acidderivatives in vivo from a renewable carbon source. More specifically, agene from Marinobacter aquaeoli coding for a hybrid fusion protein ofthe CYP153A (G307A) P450 catalytic domain, where an alanine (A) wassubstituted for a glycine (G) at position 307, was fused with a genecoding for the c-terminal FMN- and Fe/S-containing reductase domain ofP450RhF from Rhodococcus sp. NCIMB9784. The resulting polypeptide is aCYP153A-RedRhF hybrid fusion polypeptide (SEQ ID NO: 6) with acorresponding nucleic acid sequence (SEQ ID NO: 5). When thisCYP153A-reductase hybrid fusion protein was expressed in E. coli cellswith a simple carbon source such as glucose fatty acid derivatives wereefficiently converted to ω-hydroxy fatty acid derivatives (see Example1). Other examples for suitable ω-hydroxylases (EC 1.14.15.3) and theirredox partners that can be used to generate similar CYP153A-reductasehybrid fusion polypeptides are listed in Tables 2A and 2B (supra).

CYP153A-Reductase Hybrid Fusion Polypeptide Variants

The present disclosure identifies CYP153A-reductase hybrid fusionpolypeptide variants that result in high titer, yield and/orproductivity of ω-hydroxylated fatty acid derivative compositions whenexpressed in host cells. In non-limiting examples of the presentdisclosure (see Examples 1-7, infra) the hybrid CYP153A(G307A)-RedRhFfusion polypeptide (supra) was used as a template to efficientlyengineer CYP153A-reductase hybrid fusion polypeptide variants to produceincreased amounts of ω-OH fatty acids and ω-OH fatty acid derivatives.For example, such a CYP153A-reductase hybrid fusion polypeptide variantcan efficiently convert compounds such as dodecanoic acid to 12-hydroxydodecanoic acid in vivo from a simple carbon source such as glucose. Anysimple carbon source, e.g., as derived from a renewable feedstock issuitable. It was shown that an engineered CYP153A-reductase hybridfusion polypeptide variants (i.e., illustrated via an engineeredCYP153A-RedRhF hybrid fusion polypeptide variant) can efficientlyconvert fatty acids in vivo to specific desirable compounds includingω-OH fatty acids when ω-expressed with a thioesterase in a host cellsuch as E. coli by using a carbon source such as glucose from arenewable feedstock (see Examples, infra). By following the presentdisclosure, other hybrid fusion polypeptide variants can be engineeredby linking a mutated gene such as a gene coding for a CYP153A protein toa mutated gene coding for a c-terminal reductase domain (see Tables 2Athrough 2D, supra). Variations are encompassed herein, for example,mutating both genes (the P5450 and reductase domain) or mutating onegene (the P450 or reductase domain). Following these instructions,similar fusion protein variants can be created from other types ofω-hydroxylases.

Thus, the present disclosure relates to CYP153A-reductase hybrid fusionpolypeptide variants that result in high titer, yield and/orproductivity of ω-hydroxylated fatty acid derivative compositions whenexpressed in host cells. The CYP153A-reductase hybrid fusion polypeptidevariants have one or more mutations in the CYP153A domain or reductasedomain or both. In one embodiment, the present disclosure provides aCYP153A-reductase hybrid fusion polypeptide variant having at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 6 and having one or more mutation at an aminoacid position including position 27, 82, 141, 178, 231, 309, 407, 415,516, 666 and/or 796, wherein the CYP153A-reductase hybrid fusionpolypeptide variant catalyzes the conversion of a fatty acid to an ω-OHfatty acid. More specifically, the CYP153A-reductase hybrid fusionpolypeptide variant has one or more of the following mutations,including R27L where arginine (R) is substituted with lysine (L);position R82D where arginine (R) is substituted with aspartic acid (D);position V141I where valine is substituted with isoleucine (I); positionV141Q where valine (V) is substituted with glutamine (Q); position V141Gwhere valine (V) is substituted with glycine (G); position V141M wherevaline (V) is substituted with methionine (M); position V141L wherevaline (V) is substituted with leucine (L); position V141T where valine(V) is substituted with threonine (T); position R178N where arginine (R)is substituted with asparagine (N); position A231T where alanine (A) issubstituted with threonine (T); position N309R where asparagine (N) issubstituted with arginine (R); position N407A where asparagine (N) issubstituted with alanine (A); position V415R where valine (V) issubstituted with arginine (R); position T516V where threonine (T) issubstituted with valine (V); position P666A where proline (P) issubstituted with alanine (A); position P666D where proline (P) issubstituted with aspartic acid (D); and position A796V where alanine (A)is substituted with valine (V). Examples of CYP153A-reductase hybridfusion polypeptide variants include SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46. In one embodiment,the CYP153A-reductase hybrid fusion polypeptide variant is a hybridcyp153A-RedRhF-type fusion protein variant. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant in a recombinanthost cell results in a higher titer of an ω-OH fatty acid derivative orcomposition thereof as compared to the titer of an ω-OH fatty acid orcomposition thereof produced by expression of a CYP153A-reductase hybridfusion polypeptide in a corresponding host cell. In another embodiment,the CYP153A-reductase hybrid fusion polypeptide variant has a mutationat amino acid position 141, including V141I and/or V141T. Herein, theexpression of the CYP153A-reductase hybrid fusion polypeptide variantwith mutations V141I or V141T in a recombinant host cell results in ahigher titer of an ω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅,C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1),C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1)and/or C_(20:1) fatty acid, respectively, as compared to a titer of anω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉,C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1),C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fattyacid produced by expression of a CYP153A-reductase hybrid fusionpolypeptide. In one embodiment, the CYP153A-reductase hybrid fusionpolypeptide variant has mutations V141I and A231T (SEQ ID NO: 32) andproduces increased amounts of ω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃,C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1),C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1),C_(19:1) and/or C_(20:1) fatty acids when expressed in a host cell withan enzymatic function of thioesterase. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant has mutations R27L,R82D, V141M, R178N and N407A (SEQ ID NO: 34) and produces increasedamounts of ω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1),C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/orC_(20:1) fatty acids when expressed in a host cell with an enzymaticfunction of thioesterase. In another embodiment, the CYP153A-reductasehybrid fusion polypeptide variant has mutation P666A (SEQ ID NO: 36) andproduces increased amounts of ω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃,C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1),C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1),C_(19:1) and/or C_(20:1) fatty acids when expressed in a host cell withan enzymatic function of thioesterase. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant has mutation A796V(SEQ ID NO: 38) and produces increased amounts of ω-OH C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1),C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1),C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fatty acids when expressedin a host cell with an enzymatic function of thioesterase. In anotherembodiment, the CYP153A-reductase hybrid fusion polypeptide variant hasmutations A796V, P666D and T516V (SEQ ID NO: 40) and produces increasedamounts of ω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1),C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/orC_(20:1) fatty acids when expressed in a host cell with an enzymaticfunction of thioesterase. In another embodiment, the CYP153A-reductasehybrid fusion polypeptide variant has mutations V141I, A231T and A796V(SEQ ID NO: 42) and produces increased amounts of ω-OH C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1),C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1),C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fatty acids when expressedin a host cell with an enzymatic function of thioesterase. In anotherembodiment, the CYP153A-reductase hybrid fusion polypeptide variant hasmutations R27L, R82D, V141M, R178N, N407A and A796V (SEQ ID NO: 44) andproduces increased amounts of ω-OH C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃,C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_(8:1), C_(9:1), C_(10:1), C_(11:1),C_(12:1), C_(13:1), C_(14:1), C_(15:1), C_(16:1), C_(17:1), C_(18:1),C_(19:1) and/or C_(20:1) fatty acids when expressed in a host cell withan enzymatic function of thioesterase. In another embodiment, theCYP153A-reductase hybrid fusion polypeptide variant has mutations V141T,A231T and A796V (SEQ ID NO: 46) and produces increased amounts of ω-OHC₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀,C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1),C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fattyacids when expressed in a host cell with an enzymatic function ofthioesterase. In one embodiment, the variants of SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQID NO: 44 and SEQ ID NO: 46 produced increased amounts of ω-OH fattyacids or fatty acid derivatives when compared to SEQ ID NO: 6. In oneembodiment, these ω-OH fatty acids are ω-OH C_(8:0) fatty acids, ω-OHC_(10:0) fatty acids, ω-OH C_(12:0) fatty acids, ω-OH C_(14:0) fattyacids, ω-OH C_(16:0) fatty acids, ω-OH C_(18:0) fatty acids, ω-OHC_(20:0) fatty acids, ω-OH C_(8:1) fatty acids, ω-OH C_(10:1) fattyacids, ω-OH C_(12:1) fatty acids, ω-OH C_(14:1) fatty acids, ω-OHC_(16:1) fatty acids, ω-OH C_(18:1) fatty acids, ω-OH C_(20:1) fattyacids, and the like.

The disclosure identifies CYP153A-reductase hybrid fusion-relatedpolynucleotide and polypeptide variants. The CYP153A-reductase hybridfusion polypeptide variants include SEQ ID NOS: 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46. TheCYP153A-reductase hybrid fusion nucleic acid variants (DNA sequences)include SEQ ID NOS: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45 and 47. However, it will be recognized thatabsolute sequence identity to CYP153A-reductase hybrid fusionpolynucleotide variants is not necessary. For example, changes in aparticular polynucleotide sequence can be made and the encodedpolypeptide screened for activity. Such changes typically includeconservative mutations and silent mutations such as, for example,through codon optimization. Modified or mutated (i.e., mutant)polynucleotides and encoded variant polypeptides can be screened for adesired function, such as, an improved function compared to the wildtype or template polypeptide, including but not limited to increasedcatalytic activity, increased stability, or decreased inhibition (e.g.,decreased feedback inhibition), using methods known in the art. Thedisclosure identifies enzymatic activities involved in various steps(i.e., reactions) of the fatty acid biosynthetic pathways describedherein according to Enzyme Classification (EC) number, and providesexemplary polypeptides (e.g., that function as specific enzymes anddisplay specific enzyme activity) categorized by such EC numbers, andexemplary polynucleotides encoding such polypeptides. Such exemplarypolypeptides and polynucleotides, which are identified herein bySequence Identifier Numbers (SEQ ID NOs; supra), are useful forengineering fatty acid pathways in host cells such as the one shown inFIG. 1. It is to be understood, however, that polypeptides andpolynucleotides described herein are exemplary and, thus, non-limiting.The sequences of homologues of exemplary polypeptides described hereinare available to those of skill in the art using databases such as, forexample, the Entrez databases provided by the National Center forBiotechnology Information (NCBI), the ExPasy databases provided by theSwiss Institute of Bioinformatics, the BRENDA database provided by theTechnical University of Braunschweig, and the KEGG database provided bythe Bioinformatics Center of Kyoto University and University of Tokyo,all which are available on the World Wide Web.

In one embodiment, a CYP153A-reductase hybrid fusion polypeptide variantfor use in practicing the disclosure has at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28 or SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQID NO: 46. In some embodiments the CYP153A-reductase hybrid fusionpolypeptide variant is derived from a CYP153A (G307A) polypeptide fromMarinobacter aquaeolei where an alanine (A) is substituted for a glycine(G), and fused with a reductase domain of P450RhF from Rhodococcus sp.NCIMB9784. Cytochrome P450RhF is self-sufficient, displays a high degreeof substrate promiscuity and catalyzes a wide range of functionalgroups. In other embodiments, a CYP153A-reductase hybrid fusionpolypeptide variant for use in practicing the disclosure has at leastabout 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%sequence identity to SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46, and may also includeone or more substitutions which results in useful characteristics and/orproperties as described herein. In other embodiments, aCYP153A-reductase hybrid fusion polypeptide variant for use inpracticing the disclosure has at least about 100%, 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91% or 90% sequence identity to SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 orSEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46. Instill other embodiments, the P450 catalytic domain of theCYP153A-reductase hybrid fusion polypeptide variant is derived from anorganism other than Marinobacter aquaeolei. Such other organismsinclude, but are not limited to, Acinetobacter sp., Mycobacteriummarinum, Polaromonas sp., Alcanivorax borkumensis., Burkholderiafungorum, Caulobacter crescentus, Hyphomonas neptunium, Rhodopseudomonaspalustris, Sphingomonas sp., Mycobacterium sp. In still otherembodiments, the reductase domain of the CYP153A-reductase hybrid fusionpolypeptide variant is derived from an organism other than Rhodococcussp. Such other organisms include, but are not limited to, Rhodococcusequi, Acinetobacter radioresistens, Burkholderia mallei, Burkholderiamallei, Ralstonia eutropha, Cupriavidus metallidurans.

In a related embodiment, the disclosure includes a CYP153A-reductasehybrid fusion polynucleotide variant that has at least about 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identityto SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,SEQ ID NO: 43, SEQ ID NO: 45 or SEQ ID NO: 47. In some embodiments thenucleic acid sequence encodes a CYP153A-reductase hybrid fusionpolypeptide variant with one or more substitutions which results inimproved characteristics and/or properties as described herein. In yetanother related embodiment, a CYP153A-reductase hybrid fusionpolypeptide variant for use in practicing the disclosure is encoded by anucleotide sequence having at least about 100%, 99%, 98%, 97%, 96%, 95%,94%, 93%, 92%, 91% or 90% sequence identity to SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ IDNO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45 or SEQ ID NO:47. In another aspect, the disclosure relates to CYP153A-reductasehybrid fusion polypeptide variants that encompass an amino acid sequenceencoded by a nucleic acid sequence that hybridizes under stringentconditions over substantially the entire length of a nucleic acidsequence corresponding to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 41, SEQ ID NO: 43, SEQ ID NO: 45 or SEQ ID NO: 47. In someembodiments the CYP153A-reductase hybrid fusion polypeptide variant isderived from Marinobacter aquaeolei. In other embodiments, the P450hybrid fusion polypeptide is derived from Acinetobacter sp.,Mycobacterium marinum, Polaromonas sp., Alcanivorax borkumensis.,Burkholderia fungorum, Caulobacter crescentus, Hyphomonas neptunium,Rhodopseudomonas palustris, Sphingomonas sp., and Mycobacterium sp.

Additional CYP153A-Reductase Hybrid Fusion Polypeptide Variants

The disclosure identifies additional CYP153A-reductase hybridfusion-related polynucleotide and polypeptide variants, wherein avariant was used as a template. The CYP153A-reductase hybrid fusionpolypeptide variant template is based on the hybridCYP153A(G307A)-RedRhF fusion polypeptide and further includes mutationA796V (SEQ ID NO: 38). A full saturation library of cyp153A-Red450RhFfusion protein was built and screened for variants that showedimprovements over cyp153A(G307A)-Red450RhF(A796V) (SEQ ID NO: 38) (seeExample 7). Mutations G307A and A796V are beneficial mutations thatimprove ω-hydroxylase activity of cyp153A (see SEQ ID NO: 38). Theresulting CYP153A-reductase hybrid fusion polypeptide variants are shownin Table 11 (infra). These CYP153A-reductase hybrid fusion polypeptidevariants produced an increased amount of ω-hydroxy fatty acids (ω-OH FFAtiter) when compared to SEQ ID NO: 38 and include SEQ ID NOS: 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94 and 96. These CYP153A-reductase hybrid fusion polypeptidevariants may produce increased amounts of ω-OH fatty acids including C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀,C_(8:1), C_(9:1), C_(10:1), C_(11:1), C_(12:1), C_(13:1), C_(14:1),C_(15:1), C_(16:1), C_(17:1), C_(18:1), C_(19:1) and/or C_(20:1) fattyacids and/or fatty acid derivatives.

The CYP153A-reductase hybrid fusion nucleic acid variants (DNAsequences) include SEQ ID NOS: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 and 95. However,it will be recognized that absolute sequence identity toCYP153A-reductase hybrid fusion polynucleotide variants is notnecessary. For example, changes in a particular polynucleotide sequencecan be made and the encoded polypeptide screened for activity. Suchchanges typically include conservative mutations and silent mutationssuch as, for example, through codon optimization. Modified or mutated(i.e., mutant) polynucleotides and encoded variant polypeptides can bescreened for a desired function, such as, an improved function comparedto the wild type or template polypeptide, including but not limited toincreased catalytic activity, increased stability, or decreasedinhibition (e.g., decreased feedback inhibition), using methods known inthe art. The disclosure identifies enzymatic activities involved invarious steps (i.e., reactions) of the fatty acid biosynthetic pathwaysdescribed herein according to Enzyme Classification (EC) number, andprovides exemplary polypeptides (e.g., that function as specific enzymesand display specific enzyme activity) categorized by such EC numbers,and exemplary polynucleotides encoding such polypeptides. Such exemplarypolypeptides and polynucleotides, which are identified herein bySequence Identifier Numbers (SEQ ID NOs; supra), are useful forengineering fatty acid pathways in host cells such as the one shown inFIG. 1. It is to be understood, however, that polypeptides andpolynucleotides described herein are exemplary and, thus, non-limiting.The sequences of homologues of exemplary polypeptides described hereinare available to those of skill in the art using databases such as, forexample, the Entrez databases provided by the National Center forBiotechnology Information (NCBI), the ExPasy databases provided by theSwiss Institute of Bioinformatics, the BRENDA database provided by theTechnical University of Braunschweig, and the KEGG database provided bythe Bioinformatics Center of Kyoto University and University of Tokyo,all which are available on the World Wide Web.

In one embodiment, a CYP153A-reductase hybrid fusion polypeptide variantfor use in practicing the disclosure has at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ IDNO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,SEQ ID NO: 68 or SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO:76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ IDNO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94 andSEQ ID NO: 96. In some embodiments the CYP153A-reductase hybrid fusionpolypeptide variant is derived from a CYP153A (G307A) polypeptide fromMarinobacter aquaeolei where a glycine (G) is replaced with an alanine(A), and fused with a reductase domain of P450RhF from Rhodococcus sp.NCIMB9784, and includes an additional mutation of A796V where an alanine(A) is replaced with a valine (V). Cytochrome P450RhF isself-sufficient, displays a high degree of substrate promiscuity andcatalyzes a wide range of functional groups. In other embodiments, aCYP153A-reductase hybrid fusion polypeptide variant for use inpracticing the disclosure has at least about 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO:38, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ IDNO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQID NO: 66, SEQ ID NO: 68 or SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ IDNO: 94 or SEQ ID NO: 96, and may also include one or more substitutionswhich results in useful characteristics and/or properties as describedherein. In other embodiments, a CYP153A-reductase hybrid fusionpolypeptide variant for use in practicing the disclosure has at leastabout 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% sequenceidentity to SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:64, SEQ ID NO: 66, SEQ ID NO: 68 or SEQ ID NO: 70, SEQ ID NO: 72, SEQ IDNO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92,SEQ ID NO: 94 or SEQ ID NO: 96. In still other embodiments, the P450catalytic domain of the CYP153A-reductase hybrid fusion polypeptidevariant is derived from an organism other than Marinobacter aquaeolei.Such other organisms include, but are not limited to, Acinetobacter sp.,Mycobacterium marinum, Polaromonas sp., Alcanivorax borkumensis.,Burkholderia fungorum, Caulobacter crescentus, Hyphomonas neptunium,Rhodopseudomonas palustris, Sphingomonas sp., Mycobacterium sp. In stillother embodiments, the reductase domain of the CYP153A-reductase hybridfusion polypeptide variant is derived from an organism other thanRhodococcus sp. Such other organisms include, but are not limited to,Rhodococcus equi, Acinetobacter radioresistens, Burkholderia mallei,Burkholderia mallei, Ralstonia eutropha, Cupriavidus metallidurans.

In a related embodiment, the disclosure includes a CYP153A-reductasehybrid fusion polynucleotide variant that has at least about 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identityto SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ IDNO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO:83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ IDNO: 93 or SEQ ID NO: 95. In some embodiments the nucleic acid sequenceencodes a CYP153A-reductase hybrid fusion polypeptide variant with oneor more substitutions which results in improved characteristics and/orproperties as described herein. In yet another related embodiment, aCYP153A-reductase hybrid fusion polypeptide variant for use inpracticing the disclosure is encoded by a nucleotide sequence having atleast about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90%sequence identity to SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ IDNO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71,SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ IDNO: 91, SEQ ID NO: 93 or SEQ ID NO: 95. In another aspect, thedisclosure relates to CYP153A-reductase hybrid fusion polypeptidevariants that encompass an amino acid sequence encoded by a nucleic acidsequence that hybridizes under stringent conditions over substantiallythe entire length of a nucleic acid sequence corresponding to SEQ ID NO:47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75,SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93 or SEQ IDNO: 95. In some embodiments the CYP153A-reductase hybrid fusionpolypeptide variant is derived from a Marinobacter aquaeolei species. Inother embodiments, the P450 hybrid fusion polypeptide is derived fromAcinetobacter sp., Mycobacterium marinum, Polaromonas sp., Alcanivoraxborkumensis Burkholderia fungorum, Caulobacter crescentus, Hyphomonasneptunium, Rhodopseudomonas palustris, Sphingomonas sp., andMycobacterium sp.

Sequences

The variants shown in the table below are based on hybrid cytochromeP450 cyp153A16(G307A)-RedRhF fusion protein.

SEQUENCE TABLE with Variants based on Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein Sequence Identifying DescriptionNumber (SEQ ID NO) P450 Cyp153A Marinobacter aquaeolei VT8 wild typesequence (DNA) 1 P450 Cyp153A Marinobacter aquaeolei VT8 wild typesequence (protein) 2 Cytochrome P450 Cyp153A16(G307A) from Marinobacteraquaeolei, 3 YP_957888 (DNA) Cytochrome P450 Cyp153A16(G307A) fromMarinobacter aquaeolei, 4 YP_957888 (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 5 (Template) (DNA) HybridCytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 6 (Template)(protein) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein7 Variant R27L (DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhFFusion Protein 8 Variant R27L (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 9 Variant R82D (DNA) HybridCytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 10 Variant R82D(protein) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein11 Variant V141I (DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhFFusion Protein 12 Variant V141I (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 13 Variant V141Q (DNA) HybridCytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 14 Variant V141Q(protein) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein15 Variant V141G (DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhFFusion Protein 16 Variant V141G (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 17 Variant V141M (DNA) HybridCytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 18 Variant V141M(protein) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein19 Variant V141L (DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhFFusion Protein 20 Variant V141L (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 21 Variant V141T (DNA) HybridCytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 22 Variant V14IT(protein Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein23 Variant R178N (DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhFFusion Protein 24 Variant R178N (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 25 Variant N309R (DNA) HybridCytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 26 Variant N309R(protein) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein27 Variant N407A (DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhFFusion Protein 28 Variant N407A (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 29 Variant V415R (DNA) HybridCytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 30 Variant V415R(protein) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein31 Variant V141I and A231T (DNA) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 32 Variant V141I and A231T(protein) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein33 Variant R27L, R82D, V141M, R178N and N407A (DNA) Hybrid CytochromeP450 Cyp153A16(G307A)-RedRhF Fusion Protein 34 Variant R27L, R82D,V141M, R178N and N407A (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 35 Variant P666A (DNA) HybridCytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 36 Variant P666A(protein) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein37 Variant A796V (DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhFFusion Protein 38 Variant A796V (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 39 Variant T516V, P666D and A796V(DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 40Variant T516V, P666D and A796V (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 41 Variant V141I, A231T and A796V(DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 42Variant V141I, A231T and A796V (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 43 Variant R27L, R82D, V141M,R178N, N407A and A796V (DNA) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 44 Variant R27L, R82D, V141M,R178N, N407A and A796V (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF Fusion Protein 45 Variant V141T, A231T and A796V(DNA) Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF Fusion Protein 46Variant V141T, A231T and A796V (protein)

The variants shown in the table below are based on hybrid CytochromeP450 cyp153A(G307A)-Red450RhF(A796V) fusion protein.

SEQUENCE TABLE with Variants based on Hybrid Cytochrome P450cyp153A(G307A)-Red450RhF(A796V) Fusion Protein Sequence IdentifyingDescription Number (SEQ ID NO) Hybrid Cytochrome P450Cyp153A(G307A)-Red450RhF(A796V) Fusion 37 Protein (Template) (DNA)Hybrid Cytochrome P450 Cyp153A(G307A)-Red450RhF(A796V) Fusion 38 Protein(Template) (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 47 Protein Variant D747N (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 48 ProteinVariant D747N (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 49 Protein Variant Q12W (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 50 ProteinVariant Q12W (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 51 Protein Variant P327D (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 52 ProteinVariant P327D (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 53 Protein Variant R14F (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 54 ProteinVariant R14F (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 55 Protein Variant N61L (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 56 ProteinVariant N61L (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 57 Protein Variant Q28M (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 58 ProteinVariant Q28M (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 59 Protein Variant S13K (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 60 ProteinVariant S13K (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 61 Protein Variant V771F (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 62 ProteinVariant V771F (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 63 Protein Variant Q12T (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 64 ProteinVariant Q12T (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 65 Protein Variant K119R (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 66 ProteinVariant K119R (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 67 Protein Variant D10Y (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 68 ProteinVariant D10Y (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 69 Protein Variant Q12R (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 70 ProteinVariant Q12R (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 71 Protein Variant I11L (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 72 ProteinVariant I11L (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 73 Protein Variant Q28T (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 74 ProteinVariant Q28T (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 75 Protein Variant A231Y (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 76 ProteinVariant A231Y (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 77 Protein Variant P745R (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 78 ProteinVariant P745R (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 79 Protein Variant D9N (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 80 ProteinVariant D9N (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 81 Protein Variant T770G (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 82 ProteinVariant T770G (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 83 Protein Variant Y413R (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 84 ProteinVariant Y413R (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 85 Protein Variant M784I (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 86 ProteinVariant M784I (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 87 Protein Variant D9K (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 88 ProteinVariant D9K (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 89 Protein Variant E749L (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 90 ProteinVariant E749L (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 91 Protein Variant S233L (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 92 ProteinVariant S233L (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 93 Protein Variant E757A (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 94 ProteinVariant E757A (protein) Hybrid Cytochrome P450Cyp153A16(G307A)-RedRhF(A796V) Fusion 95 Protein Variant L703G (DNA)Hybrid Cytochrome P450 Cyp153A16(G307A)-RedRhF(A796V) Fusion 96 ProteinVariant L703G (protein)

Variations and Mutations

A variant polypeptide as used herein refers to a polypeptide having anamino acid sequence that differs from a wild-type or templatepolypeptide by at least one amino acid. For example, the variant (e.g.,mutant) can have one or more of the following conservative amino acidsubstitutions, including but not limited to, replacement of an aliphaticamino acid, such as alanine, valine, leucine, and isoleucine, withanother aliphatic amino acid; replacement of a serine with a threonine;replacement of a threonine with a serine; replacement of an acidicresidue, such as aspartic acid and glutamic acid, with another acidicresidue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the variant polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 99, or more amino acidsubstitutions, additions, insertions, or deletions. Some preferredfragments of a polypeptide that function as a variant or mutant retainsome or all of the biological function (e.g., enzymatic activity) of thecorresponding wild-type polypeptide. In some embodiments, the fragmentretains at least 75%, at least 80%, at least 90%, at least 95%, or atleast 98% or more of the biological function of the correspondingwild-type or template polypeptide. In other embodiments, the fragment ormutant retains about 100% of the biological function of thecorresponding wild-type or template polypeptide. In other embodiments,some fragments exhibit increased biological function as compared to thecorresponding wild-type or template polypeptide. Guidance in determiningwhich amino acid residues may be substituted, inserted, or deletedwithout affecting biological activity may be found using computerprograms well known in the art, for example, LASERGENE software(DNASTAR, Inc., Madison, Wis.). In some embodiments, a fragment exhibitsincreased biological function as compared to a corresponding wild-typepolypeptide or template polypeptide. For example, a fragment may displayat least a 10%, at least a 25%, at least a 50%, at least a 75%, or atleast a 90% improvement in enzymatic activity as compared to thecorresponding wild-type polypeptide or template polypeptide. In otherembodiments, the fragment displays at least 100%, at least 200%, or atleast 500% improvement in enzymatic activity as compared to thecorresponding wild-type polypeptide or template polypeptide.

It is understood that the polypeptides described herein may haveadditional conservative or non-essential amino acid substitutions whichdo not have a substantial effect on the polypeptide function. Whether ornot a particular substitution will be tolerated (i.e., will notadversely affect the desired biological function, such as ω-hydroxylaseenzymatic activity), can be determined as known in the art (see Bowie etal. (1990) Science, 247:1306-1310). A conservative amino acidsubstitution is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine), and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Variants can be naturally occurring or created in vitro. In particular,such variants can be created using genetic engineering techniques, suchas site directed mutagenesis, random chemical mutagenesis, ExonucleaseIII deletion procedures, or standard cloning techniques. Alternatively,such variants, mutants, fragments, analogs, or derivatives can becreated using chemical synthesis or modification procedures. Methods ofmaking variants are well known in the art. For example, variants can beprepared by using random and site-directed mutagenesis. Random andsite-directed mutagenesis are generally known in the art (see, forexample, Arnold (1993) Curr. Opin. Biotech. 4:450-455). Randommutagenesis can be achieved using error prone PCR (see, for example,Leung et al. (1989) Technique 1:11-15; and Caldwell et al. (1992) PCRMethods Applic. 2: 28-33). In error prone PCR, the actual PCR isperformed under conditions where the copying fidelity of the DNApolymerase is low, such that a high rate of point mutations is obtainedalong the entire length of the PCR product. Briefly, in such procedures,nucleic acids to be mutagenized (e.g., a polynucleotide sequenceencoding a P450 protein or P450 hybrid fusion polypeptide) are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase, and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction can be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mMKCl, 10 mM Tris HCl (pH 8.3), 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR can be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedby those in the art that these parameters can be varied as appropriate.The mutagenized nucleic acids are then cloned into an appropriatevector, and the activities of the polypeptides encoded by themutagenized nucleic acids are evaluated. Site-directed mutagenesis canbe achieved using oligonucleotide-directed mutagenesis to generatesite-specific mutations in any cloned DNA of interest. Oligonucleotidemutagenesis is described in the art (see, for example, Reidhaar-Olson etal. (1988) Science 241:53-57). Briefly, in such procedures a pluralityof double stranded oligonucleotides bearing one or more mutations to beintroduced into the cloned DNA are synthesized and inserted into thecloned DNA to be mutagenized (e.g., a polynucleotide sequence encoding aP450 polypeptide or P450 hybrid fusion polypeptide). Clones containingthe mutagenized DNA are recovered, and the activities of thepolypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction (see U.S. Pat. No. 5,965,408). Still another methodof generating variants is sexual PCR mutagenesis. In sexual PCRmutagenesis, forced homologous recombination occurs between DNAmolecules of different, but highly related, DNA sequences in vitro as aresult of random fragmentation of the DNA molecule based on sequencehomology. This is followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is describedpublications known in the art (see, for example, Stemmer (1994) Proc.Natl. Acad. Sci. U.S.A. 91:10747-10751). Variants can also be created byin vivo mutagenesis. In some embodiments, random mutations in a nucleicacid sequence are generated by propagating the sequence in a bacterialstrain, such as an E. coli strain, which carries mutations in one ormore of the DNA repair pathways. Such mutator strains have a higherrandom mutation rate than that of a wild-type strain. Propagating a DNAsequence (e.g., a polynucleotide sequence encoding an P450 hybrid fusionpolypeptide) in one of these strains will eventually generate randommutations within the DNA. Mutator strains suitable for use for in vivomutagenesis are described in publication in the art (see, for example,International Patent Application Publication No. WO1991/016427).Variants can also be generated using cassette mutagenesis. In cassettemutagenesis, a small region of a double-stranded DNA molecule isreplaced with a synthetic oligonucleotide cassette that differs from thenative sequence. The oligonucleotide often contains a completely and/orpartially randomized native sequence. Recursive ensemble mutagenesis canalso be used to generate variants. Recursive ensemble mutagenesis is analgorithm for protein engineering (i.e., protein mutagenesis) developedto produce diverse populations of phenotypically related mutants whosemembers differ in amino acid sequence. This method uses a feedbackmechanism to control successive rounds of combinatorial cassettemutagenesis (see, for example, Arkin et al. (1992) Proc. Natl. Acad.Sci., U.S.A. 89:7811-7815). In some embodiments, variants are createdusing exponential ensemble mutagenesis. Exponential ensemble mutagenesisis a process for generating combinatorial libraries with a highpercentage of unique and functional mutants, wherein small groups ofresidues are randomized in parallel to identify, at each alteredposition, amino acids which lead to functional proteins (see, forexample, Delegrave et al. (1993) Biotech. Res. 11:1548-1552). In someembodiments, variants are created using shuffling procedures whereinportions of a plurality of nucleic acids that encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences that encode chimeric polypeptides (as described in, forexample, U.S. Pat. Nos. 5,965,408 and 5,939,250).

Host Cells

Strategies to increase production of ω-OH fatty acid compositions byrecombinant host cells include increased flux through the fatty acidbiosynthetic pathway by expressing a CYP153A-reductase hybrid fusiongene and a thioesterase gene in the production host. As used herein, theterm recombinant host cell or engineered host cell refers to a host cellwhose genetic makeup has been altered relative to the correspondingwild-type host cell, for example, by deliberate introduction of newgenetic elements and/or deliberate modification of genetic elementsnaturally present in the host cell. The offspring of such recombinanthost cells also contain these new and/or modified genetic elements. Inany of the aspects of the disclosure described herein, the host cell canbe selected from a plant cell, insect cell, fungus cell (e.g., afilamentous fungus, such as Candida sp., or a budding yeast, such asSaccharomyces sp.), an algal cell and a bacterial cell. In oneembodiment, recombinant host cells are recombinant microorganisms.Examples of host cells that are microorganisms include, but are notlimited to, cells from the genus Escherichia, Bacillus, Lactobacillus,Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma,Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia,Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces,Yarrowia, or Streptomyces. In some embodiments, the host cell is aGram-positive bacterial cell. In other embodiments, the host cell is aGram-negative bacterial cell. In some embodiments, the host cell is anE. coli cell. In some embodiment, the host cell is an E. coli B cell, anE. coli C cell, an E. coli K cell, or an E. coli W cell. In otherembodiments, the host cell is a Bacillus lentus cell, a Bacillus breviscell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell,a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacilluscirculans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell,a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtiliscell, or a Bacillus amyloliquefaciens cell. In other embodiments, thehost cell is a Trichoderma koningii cell, a Trichoderma viride cell, aTrichoderma reesei cell, a Trichoderma longibrachiatum cell, anAspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillusfoetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell,an Aspergillus oryzae cell, a Humicola insolens cell, a Humicolalanuginose cell, a Rhodococcusopacus cell, a Rhizomucormiehei cell, or aMucormichei cell. In yet other embodiments, the host cell is aStreptomyces lividans cell or a Streptomyces murinus cell. In yet otherembodiments, the host cell is an Actinomycetes cell. In someembodiments, the host cell is a Saccharomyces cerevisiae cell.

In other embodiments, the host cell is a eukaryotic plant cell, an algacell, a cyanobacterium cell, a green-sulfur bacterium cell, a greennon-sulfur bacterium cell, a purple sulfur bacterium cell, a purplenon-sulfur bacterium cell, an extremophile cell, a yeast cell, a funguscell, an engineered cell of any of the organisms described herein, or asynthetic organism. In some embodiments, the host cell islight-dependent or fixes carbon. In some embodiments, the host cell hasautotrophic activity. In some embodiments, the host cell hasphotoautotrophic activity, such as in the presence of light. In someembodiments, the host cell is heterotrophic or mixotrophic in theabsence of light. In certain embodiments, the host cell is a cell fromArabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays,Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela salina,Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, SynechocystisSp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum,Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum,Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridiumljungdahlii, Clostridium thermocellum, Penicillium chrysogenum,Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pseudomonas fluorescens, or Zymomonas mobilis. In one embodiment, themicrobial cell is from a cyanobacteria including, but not limited to,Prochlorococcus, Synechococcus, Synechocystis, Cyanothece, and Nostocpunctiforme. In another embodiment, the microbial cell is from aspecific cyanobacterial species including, but not limited to,Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, andSynechococcus sp. PCC7001.

Expression Vectors

In some embodiments, a polynucleotide (or gene) sequence is provided tothe host cell by way of a recombinant vector, which includes a promoteroperably linked to the polynucleotide sequence. In certain embodiments,the promoter is a developmentally-regulated, an organelle-specific, atissue-specific, an inducible, a constitutive, or a cell-specificpromoter. In some embodiments, the recombinant vector includes at leastone sequence selected from an expression control sequence operativelycoupled to the polynucleotide sequence; a selection marker operativelycoupled to the polynucleotide sequence; a marker sequence operativelycoupled to the polynucleotide sequence; a purification moietyoperatively coupled to the polynucleotide sequence; a secretion sequenceoperatively coupled to the polynucleotide sequence; and a targetingsequence operatively coupled to the polynucleotide sequence. Theexpression vectors described herein include a polynucleotide sequence ina form suitable for expression of the polynucleotide sequence in a hostcell. It will be appreciated by those skilled in the art that the designof the expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of polypeptidedesired, and the like. The expression vectors described herein can beintroduced into host cells to produce polypeptides, including fusionpolypeptides, encoded by the polynucleotide sequences as described above(supra). Expression of genes encoding polypeptides in prokaryotes, forexample, E. coli, is most often carried out with vectors containingconstitutive or inducible promoters directing the expression of eitherfusion or non-fusion polypeptides. Fusion vectors add a number of aminoacids to a polypeptide encoded therein, usually to the amino- orcarboxy-terminus of the recombinant polypeptide. Such fusion vectorstypically serve one or more of the following three purposes includingincreasing expression of the recombinant polypeptide; increasing thesolubility of the recombinant polypeptide; and aiding in thepurification of the recombinant polypeptide by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant polypeptide. This allows separation of therecombinant polypeptide from the fusion moiety after purification of thefusion polypeptide. Examples of such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin, and enterokinase.Exemplary fusion expression vectors include pGEX vector (PharmaciaBiotech, Inc., Piscataway, N.J.; Smith et al. (1988) Gene 67:31-40),pMAL vector (New England Biolabs, Beverly, Mass.), and pRITS vector(Pharmacia Biotech, Inc., Piscataway, N.J.), which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant polypeptide.

Examples of inducible, non-fusion E. coli expression vectors includepTrc vector (Amann et al. (1988) Gene 69:301-315) and pET 11d vector(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains such as BL21(DE3) or HMS174(DE3)from a resident λ prophage harboring a T7 gn1 gene under thetranscriptional control of the lacUV 5 promoter. Suitable expressionsystems for both prokaryotic and eukaryotic cells are well known in theart (see, e.g., Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, second edition, Cold Spring Harbor Laboratory). Examples ofinducible, non-fusion E. coli expression vectors include pTrc vector(Amann et al. (1988) Gene 69:301-315) and PET 11d vector (Studier et al.(1990) Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif., pp. 60-89). In certain embodiments, apolynucleotide sequence of the disclosure is operably linked to apromoter derived from bacteriophage T5. In one embodiment, the host cellis a yeast cell. In this embodiment, the expression vector is a yeastexpression vector. Vectors can be introduced into prokaryotic oreukaryotic cells via a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a host cell. Suitablemethods for transforming or transfecting host cells can be found in, forexample, Sambrook et al. (supra). For stable transformation of bacterialcells, it is known that (depending upon the expression vector andtransformation technique used) a certain fraction of cells will take-upand replicate the expression vector. In order to identify and selectthese transformants, a gene that encodes a selectable marker (e.g.,resistance to an antibiotic) can be introduced into the host cells alongwith the gene of interest. Selectable markers include those that conferresistance to drugs such as, but not limited to, ampicillin, kanamycin,chloramphenicol, or tetracycline. Nucleic acids encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding a polypeptide described herein or can be introduced on aseparate vector.

Optional Pathway Engineering

The host cells or microorganisms of the disclosure include host strainsor host cells that are genetically engineered or modified to containalterations in order to test the efficiency of specific mutations onenzymatic activities (i.e., recombinant cells or microorganisms).Various optional genetic manipulations and alterations can be usedinterchangeably from one host cell to another, depending on what nativeenzymatic pathways are present in the original host cell. In oneembodiment, a host strain can be used for testing the expression of aCYP153A-reductase hybrid fusion polypeptide variant in combination withother biosynthetic polypeptides (e.g., enzymes). A host strain mayencompasses a number of genetic alterations in order to test specificvariables, including but not limited to, culture conditions includingfermentation components, carbon source (e.g., feedstock), temperature,pressure, reduced culture contamination conditions, and oxygen levels.

In one embodiment, a host strain encompasses an optional fadE and fhuAdeletion. Acyl-CoA dehydrogenase (FadE) is an enzyme that is importantfor metabolizing fatty acids. It catalyzes the second step in fatty acidutilization (beta-oxidation), which is the process of breaking longchains of fatty acids (acyl-CoAs) into acetyl-CoA molecules. Morespecifically, the second step of the β-oxidation cycle of fatty aciddegradation in bacteria is the oxidation of acyl-CoA to 2-enoyl-CoA,which is catalyzed by FadE. When E. coli lacks FadE, it cannot grow onfatty acids as a carbon source but it can grow on acetate. The inabilityto utilize fatty acids of any chain length is consistent with thereported phenotype of fadE strains, i.e., fadE mutant strains where FadEfunction is disrupted. The fadE gene can be optionally knocked out orattenuated to assure that acyl-CoAs, which may be intermediates in afatty acid derivative pathway, can accumulate in the cell such that allacyl-CoAs can be efficiently converted to fatty acid derivatives.However, fadE attenuation is optional when sugar is used as a carbonsource since under such condition expression of FadE is likely repressedand FadE therefore may only be present in small amounts and not able toefficiently compete with ester synthase or other enzymes for acyl-CoAsubstrates. FadE is repressed due to catabolite repression. E. coli andmany other microbes prefer to consume sugar over fatty acids, so whenboth sources are available sugar is consumed first by repressing the fadregulon (see D. Clark, J Bacteriol. (1981) 148(2):521-6)). Moreover, theabsence of sugars and the presence of fatty acids induces FadEexpression. Acyl-CoA intermediates could be lost to the beta oxidationpathway since the proteins expressed by the fad regulon (including FadE)are up-regulated and will efficiently compete for acyl-CoAs. Thus, itcan be beneficial to have the fadE gene knocked out or attenuated. Sincemost carbon sources are mainly sugar based, it is optional to attenuateFadE. The gene fhuA codes for the TonA protein, which is anenergy-coupled transporter and receptor in the outer membrane of E. coli(V. Braun (2009) J Bacteriol. 191(11):3431-3436). Its deletion isoptional. The fhuA deletion allows the cell to become more resistant tophage attack which can be beneficial in certain fermentation conditions.Thus, it may be desirable to delete fhuA in a host cell that is likelysubject to potential contamination during fermentation runs.

In another embodiment, the host strain (supra) also encompasses optionaloverexpression of one or more of the following genes including fadR,fabA, fabD, fabG, fabH, fabV, and/or fabF. Examples of such genes arefadR from Escherichia coli, fabA from Salmonella typhimurium(NP_460041), fabD from Salmonella typhimurium (NP_460164), fabG fromSalmonella typhimurium (NP_460165), fabH from Salmonella typhimurium(NP_460163), fabV from Vibrio cholera (YP_001217283), and fabF fromClostridium acetobutylicum (NP_350156). The overexpression of one ormore of these genes, which code for enzymes and regulators in fatty acidbiosynthesis, can serve to increase the titer of fatty-acid derivativecompounds including ω-OH fatty acids and derivatives thereof undervarious culture conditions.

In another embodiment, E. coli strains are used as host cells for theproduction of ω-OH fatty acids and derivatives thereof. Similarly, thesehost cells provide optional overexpression of one or more biosynthesisgenes (i.e., genes coding for enzymes and regulators of fatty acidbiosynthesis) that can further increase or enhance the titer offatty-acid derivative compounds such as fatty acid derivatives (e.g.,ω-OH fatty acids and α,ω-diacids, etc.) under various culture conditionsincluding, but not limited to, fadR, fabA, fabD, fabG, fabH, fabV and/orfabF. Examples of genetic alterations include fadR from Escherichiacoli, fabA from Salmonella typhimurium (NP_460041), fabD from Salmonellatyphimurium (NP_460164), fabG from Salmonella typhimurium (NP_460165),fabH from Salmonella typhimurium (NP_460163), fabV from Vibrio cholera(YP_001217283), and fabF from Clostridium acetobutylicum (NP_350156). Insome embodiments, synthetic operons that carry these biosynthetic genescan be engineered and expressed in cells in order to test P450expression under various culture conditions and/or further enhance ω-OHfatty acid and α,ω-diacid production. Such synthetic operons contain oneor more biosynthetic gene. An engineered operon may contain optionalfatty acid biosynthetic genes, including fabV from Vibrio cholera, fabHfrom Salmonella typhimurium, fabD from S. typhimurium, fabG from S.typhimurium, fabA from S. typhimurium and/or fabF from Clostridiumacetobutylicum that may be used to facilitate overexpression of fattyacid derivatives in order to test specific culture conditions. Oneadvantage of such synthetic operons is that the rate of ω-OH fatty acidderivative production may be further increased or enhanced.

In some embodiments, the host cells or microorganisms that are used toexpress ACP and biosynthetic enzymes (e.g., ω-hydroxylase, thioesterase,etc.) will further express genes that encompass certain enzymaticactivities that can increase the production to one or more particularfatty acid derivative(s) such as ω-OH fatty acids, ω-OH fatty acidderivatives, α,ω-diacids and the like. In one embodiment, the host cellhas thioesterase activity (E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C.3.1.1.5) for the production of fatty acids which can be increased byoverexpressing the gene. In another embodiment, the host cell has estersynthase activity (E.C. 2.3.1.75) for the production of fatty esters. Inanother embodiment, the host cell has acyl-ACP reductase (AAR) (E.C.1.2.1.80) activity and/or alcohol dehydrogenase activity (E.C. 1.1.1.1.)and/or fatty alcohol acyl-CoA reductase (FAR) (E.C. 1.1.1.*) activityand/or carboxylic acid reductase (CAR) (EC 1.2.99.6) activity for theproduction of fatty alcohols. In another embodiment, the host cell hasacyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity for the production offatty aldehydes. In another embodiment, the host cell has acyl-ACPreductase (AAR) (E.C. 1.2.1.80) activity and decarbonylase (ADC)activity for the production of alkanes and alkenes. In anotherembodiment, the host cell has acyl-CoA reductase (E.C. 1.2.1.50)activity, acyl-CoA synthase (FadD) (E.C. 2.3.1.86) activity, andthioesterase (E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5) activityfor the production of fatty alcohols. In another embodiment, the hostcell has ester synthase activity (E.C. 2.3.1.75), acyl-CoA synthase(FadD) (E.C. 2.3.1.86) activity, and thioesterase (E.C. 3.1.2.* or E.C.3.1. 2.14 or E.C. 3.1.1.5) activity for the production of fatty esters.In another embodiment, the host cell has OleA activity for theproduction of ketones. In another embodiment, the host cell has OleBCDactivity for the production of internal olefins. In another embodiment,the host cell has acyl-ACP reductase (AAR) (E.C. 1.2.1.80) activity andalcohol dehydrogenase activity (E.C. 1.1.1.1.) for the production offatty alcohols. In another embodiment, the host cell has thioesterase(E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5) activity anddecarboxylase activity for making terminal olefins. The expression ofenzymatic activities in microorganisms and microbial cells is taught byU.S. Pat. Nos. 8,097,439; 8,110,093; 8,110,670; 8,183,028; 8,268,599;8,283,143; 8,232,924; 8,372,610; and 8,530,221, which are incorporatedherein by reference. In other embodiments, the host cells ormicroorganisms that are used to express ACP and other biosyntheticenzymes will include certain native enzyme activities that areupregulated or overexpressed in order to produce one or more particularfatty acid derivative(s) such as ω-OH fatty acids, ω-OH fatty acidderivatives, and α,ω-diacids. In one embodiment, the host cell has anative thioesterase (E.C. 3.1.2.* or E.C. 3.1. 2.14 or E.C. 3.1.1.5)activity for the production of fatty acids which can be increased byoverexpressing the thioesterase gene.

The present disclosure includes host strains or microorganisms thatexpress genes that code for CYP153A-reductase hybrid fusion polypeptidevariants and other biosynthetic enzymes (supra). The recombinant hostcells produce fatty acid derivatives such as ω-OH fatty acids, ω-OHfatty acid derivatives, α,ω-diacids and compositions and blends thereof.The fatty acid derivatives are typically recovered from the culturemedium and/or are isolated from the host cells. In one embodiment, thefatty acid derivatives are recovered from the culture medium(extracellular). In another embodiment, the fatty acid derivatives areisolated from the host cells (intracellular). In another embodiment, thefatty acid derivatives are recovered from the culture medium andisolated from the host cells. The fatty acid derivatives or compositionsproduced by a host cell can be analyzed using methods known in the art,for example, GC-FID, in order to determine the distribution ofparticular fatty acid derivatives as well as chain lengths and degree ofsaturation of the components of the ω-OH fatty acid derivatives such asω-OH fatty acids, ω-OH fatty esters, α,ω-diacids, and the like.

Culture and Fermentation

As used herein, the term fermentation broadly refers to the conversionof organic materials into target substances by host cells, for example,the conversion of a carbon source by recombinant host cells into ω-OHfatty acids or derivatives thereof by propagating a culture of therecombinant host cells in a media comprising the carbon source. Theconditions permissive for the production refer to any conditions thatallow a host cell to produce a desired product, such as ω-OH fattyacids. Similarly, the condition or conditions in which thepolynucleotide sequence of a vector is expressed means any conditionsthat allow a host cell to synthesize a polypeptide. Suitable conditionsinclude, for example, fermentation conditions. Fermentation conditionscan include many parameters including, but not limited to, temperatureranges, levels of aeration, feed rates and media composition. Each ofthese conditions, individually and in combination, allows the host cellto grow. Fermentation can be aerobic, anaerobic, or variations thereof(such as micro-aerobic). Exemplary culture media include broths or gels.Generally, the medium includes a carbon source that can be metabolizedby a host cell directly. In addition, enzymes can be used in the mediumto facilitate the mobilization (e.g., the depolymerization of starch orcellulose to fermentable sugars) and subsequent metabolism of the carbonsource.

For small scale production, the engineered host cells can be grown inbatches of, for example, about 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 1mL, 5 mL, 10 mL, 15 mL, 25 mL, 50 mL, 75 mL, 100 mL, 500 mL, 1 L, 2 L, 5L, or 10 L; fermented; and induced to express a desired polynucleotidesequence, such as a polynucleotide sequence encoding a P450 hybridfusion polypeptide. For large scale production, the engineered hostcells can be grown in batches of about 10 L, 100 L, 1000 L, 10,000 L,100,000 L, and 1,000,000 L or larger; fermented; and induced to expressa desired polynucleotide sequence. Alternatively, large scale fed-batchfermentation may be carried out. The ω-OH fatty acids, derivatives andcompositions thereof as described herein are found in the extracellularenvironment of the recombinant host cell culture and can be readilyisolated from the culture medium. An ω-OH fatty acid or derivativethereof may be secreted by the recombinant host cell, transported intothe extracellular environment or passively transferred into theextracellular environment of the recombinant host cell culture. The ω-OHfatty acids or derivatives thereof are isolated from a recombinant hostcell culture using routine methods known in the art.

Products Derived from Recombinant Host Cells

As used herein, the fraction of modem carbon or fM has the same meaningas defined by National Institute of Standards and Technology (NIST)Standard Reference Materials (SRMs4990B and 4990C, known as oxalic acidsstandards HOxI and HOxII, respectively. The fundamental definitionrelates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD1950). This is roughly equivalent to decay-corrected pre-IndustrialRevolution wood. For the current living biosphere (plant material), fMis approximately 1.1. Bioproducts (e.g., the fatty acid derivativesincluding ω-OH fatty acids and derivatives produced in accordance withthe present disclosure) include biologically produced organic compounds.In particular, the fatty acid derivatives (e.g., ω-OH fatty acids andderivatives thereof) produced using the fatty acid biosynthetic pathwayherein, have not been produced from renewable sources and, as such, arenew compositions of matter. These new bioproducts can be distinguishedfrom organic compounds derived from petrochemical carbon on the basis ofdual carbon-isotopic fingerprinting or ¹⁴C dating. Additionally, thespecific source of biosourced carbon (e.g., glucose vs. glycerol) can bedetermined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat.No. 7,169,588). The ability to distinguish bioproducts from petroleumbased organic compounds is beneficial in tracking these materials incommerce. For example, organic compounds or chemicals including bothbiologically based and petroleum based carbon isotope profiles may bedistinguished from organic compounds and chemicals made only ofpetroleum based materials. Hence, the bioproducts herein can be followedor tracked in commerce on the basis of their unique carbon isotopeprofile. Bioproducts can be distinguished from petroleum based organiccompounds by comparing the stable carbon isotope ratio (¹³C/¹²C) in eachsample. The ¹³C/¹²C ratio in a given bioproduct is a consequence of the¹³C/¹²C ratio in atmospheric carbon dioxide at the time the carbondioxide is fixed. It also reflects the precise metabolic pathway.Regional variations also occur. Petroleum, C3 plants (the broadleaf), C4plants (the grasses), and marine carbonates all show significantdifferences in ¹³C/¹²C and the corresponding δ¹³C values. Furthermore,lipid matter of C3 and C4 plants analyze differently than materialsderived from the carbohydrate components of the same plants as aconsequence of the metabolic pathway. Within the precision ofmeasurement, ¹³C shows large variations due to isotopic fractionationeffects, the most significant of which for bioproducts is thephotosynthetic mechanism. The major cause of differences in the carbonisotope ratio in plants is closely associated with differences in thepathway of photosynthetic carbon metabolism in the plants, particularlythe reaction occurring during the primary carboxylation (i.e., theinitial fixation of atmospheric CO₂). Two large classes of vegetationare those that incorporate the C3 (or Calvin-Benson) photosyntheticcycle and those that incorporate the C4 (or Hatch-Slack) photosyntheticcycle. In C3 plants, the primary CO₂ fixation or carboxylation reactioninvolves the enzyme ribulose-1,5-diphosphate carboxylase, and the firststable product is a 3-carbon compound. C3 plants, such as hardwoods andconifers, are dominant in the temperate climate zones. In C4 plants, anadditional carboxylation reaction involving another enzyme,phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction.The first stable carbon compound is a 4-carbon acid that is subsequentlydecarboxylated. The CO₂ thus released is refixed by the C3 cycle.Examples of C4 plants are tropical grasses, corn, and sugar cane. BothC4 and C3 plants exhibit a range of ¹³C/¹²C isotopic ratios, but typicalvalues are about −7 to about −13 per mil for C4 plants and about −19 toabout −27 per mil for C3 plants (see, e.g., Stuiver et al. (1977)Radiocarbon 19:355). Coal and petroleum fall generally in this latterrange. The ¹³C measurement scale was originally defined by a zero set byPee Dee Belemnite (PDB) limestone, where values are given in parts perthousand deviations from this material. The δ13C values are expressed inparts per thousand (per mil), abbreviated, ‰, and are calculated asfollows:δ¹3C (‰)=[(¹³C/¹²C) sample−(¹³C/¹²C) standard]/(¹³C/¹²C) standard×1000

Since the PDB reference material (RM) has been exhausted, a series ofalternative RMs have been developed in cooperation with the IAEA, USGS,NIST, and other selected international isotope laboratories. Notationsfor the per mil deviations from PDB is δ¹³C. Measurements are made onCO₂ by high precision stable ratio mass spectrometry (IRMS) on molecularions of masses 44, 45, and 46. The compositions described herein includebioproducts produced by any of the methods described herein, including,for example, fatty acid derivative products. Specifically, thebioproduct can have a δ¹³C of about −28 or greater, about −27 orgreater, −20 or greater, −18 or greater, −15 or greater, −13 or greater,−10 or greater, or −8 or greater. For example, the bioproduct can have aδ¹³C of about −30 to about −15, about −27 to about −19, about −25 toabout −21, about −15 to about −5, about −13 to about −7, or about −13 toabout −10. In other instances, the bioproduct can have a δ¹³C of about−10, −11, −12, or −12.3. Bioproducts produced in accordance with thedisclosure herein, can also be distinguished from petroleum basedorganic compounds by comparing the amount of ¹⁴C in each compound.Because ¹⁴C has a nuclear half-life of 5730 years, petroleum based fuelscontaining older carbon can be distinguished from bioproducts whichcontain newer carbon (see, e.g., Currie, Source Apportionment ofAtmospheric Particles, Characterization of Environmental Particles, J.Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPACEnvironmental Analytical Chemistry Series (Lewis Publishers, Inc.) 3-74,(1992)). The basic assumption in radiocarbon dating is that theconstancy of ¹⁴C concentration in the atmosphere leads to the constancyof ¹⁴C in living organisms. However, because of atmospheric nucleartesting since 1950 and the burning of fossil fuel since 1850, ¹⁴C hasacquired a second, geochemical time characteristic. Its concentration inatmospheric CO₂, and hence in the living biosphere, approximatelydoubled at the peak of nuclear testing, in the mid-1960s. It has sincebeen gradually returning to the steady-state cosmogenic (atmospheric)baseline isotope rate (¹⁴C/¹²C) of about 1.2×10⁻¹², with an approximaterelaxation “half-life” of 7-10 years. This latter half-life must not betaken literally; rather, one must use the detailed atmospheric nuclearinput/decay function to trace the variation of atmospheric andbiospheric ¹⁴C since the onset of the nuclear age. It is this latterbiospheric ¹⁴C time characteristic that holds out the promise of annualdating of recent biospheric carbon. ¹⁴C can be measured by acceleratormass spectrometry (AMS), with results given in units of fraction ofmodern carbon (fM). fM is defined by National Institute of Standards andTechnology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C.As used herein, fraction of modern carbon or fM has the same meaning asdefined by National Institute of Standards and Technology (NIST)Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalicacids standards HOxI and HOxII, respectively. The fundamental definitionrelates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD1950). This is roughly equivalent to decay-corrected pre-IndustrialRevolution wood. For the current living biosphere (plant material), fMis approximately 1.1. The compositions described herein includebioproducts that can have an fM¹⁴C of at least about 1. For example, thebioproduct of the disclosure can have an fM¹⁴C of at least about 1.01,an fM¹⁴C of about 1 to about 1.5, an fM¹⁴C of about 1.04 to about 1.18,or an fM¹⁴C of about 1.111 to about 1.124.

Another measurement of ¹⁴C is known as the percent of modern carbon(pMC). For an archaeologist or geologist using ¹⁴C dates, AD 1950 equalszero years old. This also represents 100 pMC. Bomb carbon in theatmosphere reached almost twice the normal level in 1963 at the peak ofthermo-nuclear weapons. Its distribution within the atmosphere has beenapproximated since its appearance, showing values that are greater than100 pMC for plants and animals living since AD 1950. It has graduallydecreased over time with today's value being near 107.5 pMC. This meansthat a fresh biomass material, such as corn, would give a ¹⁴C signaturenear 107.5 pMC. Petroleum based compounds will have a pMC value of zero.Combining fossil carbon with present day carbon will result in adilution of the present day pMC content. By presuming 107.5 pMCrepresents the ¹⁴C content of present day biomass materials and 0 pMCrepresents the ¹⁴C content of petroleum based products, the measured pMCvalue for that material will reflect the proportions of the twocomponent types. For example, a material derived 100% from present daysoybeans would give a radiocarbon signature near 107.5 pMC. If thatmaterial was diluted 50% with petroleum based products, it would give aradiocarbon signature of approximately 54 pMC. A biologically basedcarbon content is derived by assigning 100% equal to 107.5 pMC and 0%equal to 0 pMC. For example, a sample measuring 99 pMC will give anequivalent biologically based carbon content of 93%. This value isreferred to as the mean biologically based carbon result and assumes allthe components within the analyzed material originated either frompresent day biological material or petroleum based material. Abioproduct comprising one or more fatty acid derivatives as describedherein can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95,96, 97, 98, 99, or 100. In other instances, a fatty acid derivativedescribed herein can have a pMC of between about 50 and about 100; about60 and about 100; about 70 and about 100; about 80 and about 100; about85 and about 100; about 87 and about 98; or about 90 and about 95. Inyet other instances, a fatty acid derivative described herein can have apMC of about 90, 91, 92, 93, 94, or 94.2.

EXAMPLES

The following specific examples are intended to illustrate thedisclosure and should not be construed as limiting the scope of theclaims.

Protocols and Methods

Screening a Library

All protocols described herein rely on a 96 well plate—master block—2 mLsystem (Greiner Bio-One, Monroe, N.C. or Corning, Amsterdam, TheNetherlands) for growing cultures, and plates (Costar, Inc.) forextracting fatty acid species from the culture broth. The protocolsprovided below are examples of fermentation conditions. Alternativeprotocols can be used to evaluate fatty acid species production.

32° C. Plim Culture Protocol

30 μL LB culture (from an LB culture growing in a 96 well plate) wasused to inoculate 290 μL Plim media (Table 2), which was then incubatedfor approximately 16 hours at 32° C. shaking. 40 μL of the overnightseed was used to inoculate 360 μL Plim media. After growing at 32° C.for 2 hours, the cultures were induced with IPTG (final concentration 1mM) (Table 3 below). The cultures were then incubated at 32° C. withshaking for 20 hours if not noted otherwise, after which they wereextracted following the standard extraction protocol detailed below.

35° C. Nlim Culture Protocol

40 μL LB culture (from an LB culture growing in a 96 well plate) wasused to inoculate 360 μL LB media (Table 3 below), which was thenincubated for approximately 4 hours at 32° C. shaking. 40 μL of the LBseed was used to inoculate 360 μL Nlim media. After growing at 32° C.for 2 hours at 35° C., the cultures were induced with IPTG (finalconcentration 1 mM) (Table 3 below). The cultures were then incubated at35° C. with shaking for 20 hours if not noted otherwise, after whichthey were extracted following the standard extraction protocol detailedbelow.

TABLE 3 Media Names and Formulations Media Name Formulation Plim 1 x 5xPlim Salt Soln, with (NH4)₂SO₄ 1 x 1000x Trace Vitamins 1 mg/L 10 mg/mLThiamine 1 mM 1M MgSO4 0.1 mM 1M CaCl2 40 g/L 500 g/L glucose 1 x 1000xTrace minerals 10 mg/L 10 g/L Fe Citrate 100 μg/mL 100 mg/mlspectinomycin 100 mM 2M BisTris (pH 7.0) 0.5 mM Aminolevulinic acid Nlim1 x 5x Salt Soln, with NH4Cl 1 x 1000x Trace Vitamins 1 mg/L 10 mg/mLThiamine 1 mM 1M MgSO4 0.1 mM 1M CaCl2 40 g/L 500 g/L glucose 1 x 1000xTrace minerals 10 mg/L 10 g/L Fe Citrate 100 μg/mL 100 mg/mlspectinomycin 100 mM 2M BisTris (pH 7.0) 0.5 mM Aminolevulinic acid

Fatty Acid Species Standard Extraction Protocol

To each well to be extracted 80 μL of 1M HCl, followed by 400 μL ofbutyl acetate (with 500 mg/L pentadecanol as internal standard) wasadded. The 96 well plates were then heat-sealed using a plate sealer(ALPS-300 heater; Abgene, ThermoScientific, Rockford, Ill.), and shakenfor 15 minutes at 2000 rpm using MIXMATE mixer (Eppendorf, Hamburg,Germany). After shaking, the plates were centrifuged for 10 minutes at4500 rpm at room temperature (Allegra X-15R, rotor SX4750A, BeckmanCoulter, Brea, Calif.) to separate the aqueous and organic layers. 100μL of the organic layer was transferred to a 96 well plate(polypropylene, Corning, Amsterdam, The Netherlands) and derivatizedwith 100 uL of BSTFA. The plate was subsequently heat sealed and storedat −20° C. until evaluated by GC-FID using the w-OH FFA method wascarried out as follows: 1 μL of sample was injected onto an analyticalcolumn (DB-1, 10 m×180 μm×0.2 μM film thickness, available from JW121-101A) in an Agilent 7890A GC Ultra device (Agilent, Santa Clara,Calif.) with a flame ionization detector (FID) with a 1-20 split. Theinstrument was set up to detect and quantify C₁₀ to C₁₈ fatty acids andω-hydroxylated fatty acids. The protocol detailed above representsstandard conditions, which may be modified as necessary to optimize theanalytical results.

Building Error Prone Libraries

Standard techniques known to those of skill in the art were used toprepare error prone libraries. In one example, the vector backbone wasprepared using restriction endonucleases in the vector, while thecreation of diversity in the DNA insert was generated by PCRamplification from a DNA template under conditions favoring theincorporation of mismatched nucleotides. In one approach, the cloning ofthe vector backbone and a DNA insert with diversity was performed usingthe INFUSION Cloning System (Clontech Laboratories, Inc., Mountain View,Calif.), according to the manufacturer's protocol.

Building Saturation Libraries

Standard techniques known to those of skill in the art were used toprepare saturation libraries. In one example, the vector backbone wasprepared using restriction endonucleases in the vector, while thecreation of diversity in the DNA insert was generated using degenerateprimers. In one approach, the cloning of the vector backbone and a DNAinsert with diversity was performed using INFUSION Cloning System(Clontech Laboratories, Inc., Mountain View, Calif.) according to themanufacturer's protocol.

Building Combination Libraries

Mutations identified as beneficial were combined to provideCYP153-reductase hybrid fusion polypeptide variants (e.g., hybridCYP153A-RedRhF protein variants) with further improvements in theproduction of ω-OH fatty acid derivative species. Standard techniquesknown to those of skill in the art were used to prepare the combinationlibraries. In one example, the vector backbone was prepared usingrestriction endonucleases in the vector, while the creation of diversityin the DNA insert was generated using primers to introduce the desiredmutations. As described above, in one approach, the cloning of thevector backbone and a DNA insert with diversity was performed usingINFUSION Cloning System (Clontech Laboratories, Inc., Mountain View,Calif.), according to manufacturer's protocol. Combination libraries canbe generated using the transfer PCR (tPCR) protocol (Erijman et al.(2011) J. Structural Bio. 175:171-177).

Library Screening

Once the library diversity was generated in an error-prone, saturationlibrary or combination library, it was screened using one of the methodsdescribed above. Two types of hits were identified: (1) increased amountof ω-hydroxy fatty acids (ω-OH FFA titer); and/or (2) increasedconversion of fatty acids to ω-hydroxy fatty acids. The mutations in thehybrid cyp153A-RedRhF protein variants within each hit were identifiedby sequencing, using standard techniques routinely employed by those ofskill in the art. Tables 5, 6 and 7 below list the mutations (hits)identified as beneficial in saturation libraries.

Example 1: Strain and Plasmid Construction for Library Screening

This example describes the strains and plasmids constructed forsaturation or combinatorial mutagenesis library screening.

A gene coding for a hybrid-fusion protein made of the CYP153A(G307A)P450 catalytic protein from Marinobacter aquaeoli and the c-terminalFMN- and Fe/S-containing reductase domain of P450RhF from Rhodococcussp. NCIMB9784 was created as follows: The cyp165A(G307A)_Maqu gene wasamplified from genomic DNA and fused with a codon-optimized syntheticP450RhF reductase domain by cross-over PCR. The resulting fusion gene(SEQ ID NO: 5) was cloned into a pACYC-derivative (i.e., p15A replicon,kanamycin resistance marker) such that its transcription was controlledby the IPTG-inducible Ptrc promoter. The plasmid was named pEP125 (seeTable 4, infra).

The gene coding for the hybrid cyp153A(G307A)-Red450RhF fusion proteinwas also amplified from pEP125 and cloned into a pCL1920-derivativevector (SC101 replicon, spectinomycin resistance marker), such that itstranscription was controlled by the IPTG-inducible Ptrc promoter and itformed an operon with genes coding for a plant thioesterase (fatB1), avariant of 3-keto-acyl-ACP synthase (fabB) and a transcriptionalregulator (fadR). The plasmid was named pLC81 (see Table 4, infra).

Additional plasmids were created as follows: The gene coding a plantthioesterase (fatB1) from Umbellularia californica was synthesized ascodon-optimized DNA and cloned into a pCL1920-derivative vector (SC101replicon, spectinomycin resistance marker), such that its transcriptionwas controlled by the IPTG-inducible Ptrc promoter and it formed anoperon with genes coding for acetyl-CoA carboxylase (accDACB), biotinligase (birA) and a acyl-carrier protein. The plasmid was named pNH305(see Table 4, infra). Plasmid pAS033 was created by replacing fatB1 inpNH305 with a codon-optimized synthetic plant thioesterase (fatA3) fromArabidopsis thaliana (see Table 4, infra). Plasmid pEP146 was created byreplacing fatB1 in pLC81 with a codon-optimized synthetic plantthioesterase (fatA3) from Arabidopsis thaliana (see Table 4, infra).pEP146 also carried a mutation in the plasmid encoded repA protein.

Base strains used for plasmid transformation were GLP077 and BZ128.Briefly, the genome of base strain GLPH077 was manipulated as follows:the acyl-CoA dehydrogenase (fadE) gene was deleted and a transcriptionalregulator (fadR) and a synthetic fatty acid biosynthesis operon wereoverexpressed. Briefly, the genome of base strain BZ128 was manipulatedas follows: the fadE (acyl-CoA dehydrogenase) gene was deleted and asynthetic fatty acid biosynthesis operon, a β-hydroxy fatty acyl-ACPdehydratase (fabZ) and a variant of a thioesterase (tesA) wereoverexpressed. In addition, the strain had previously been subjected totransposon as well as N-methyl-N′-nitro-N-nitrosoguanidine (NTG)mutagenesis and screening.

TABLE 4 Plasmids used for library screening Plasmid Description pAS033pCL-fatA3_Atal-accDCBAbirA_Cglu-acp_Ecol pEP125pACYC-cyp153A(G307A)_Maqu-RedRhF_Rhod pNH305pCL-fatB1_Ucal-accDCBAbirA_Cglu-acp_Ecol pLC81 pCL-cyp153A(G307A)_Maqu-RedRhF_Rhod- fatB1_Ucal-fadB_Ecol-fadR_Ecol pEP146pCL*- cyp153A(G307A)_Maqu-RedRhF_Rhod- fatA3-Atal-fadB_Ecol-fadR_Ecol

The hybrid cyp153A(G307A)-Red450RhF fusion protein was tested to see ifexpression in host cells could produce ω-OH fatty acid derivatives. Amicroorganism expressing SEQ ID NO: 5 was capable of producing over a 1g/L of ω-OH fatty acid derivatives from glucose. Thus, this engineeredenzyme was selected for further evolution studies.

Example 2: Saturation Libraries of the P450 Catalytic Domain ofcyp153A(G307A)-Red450RhF Fusion Protein

A full saturation library of the P450 catalytic domain ofcyp153A-Red450RhF fusion protein, was built and screened for variantsthat showed improvements over cyp153A(G307A)-Red450RhF (i.e., thetemplate polypeptide). G307A (i.e., an alanine residue replaced aglycine in position 307) is a beneficial mutation that improvesω-hydroxylase activity of cyp153A (see Honda Malca et al. (2012) Chem.Commun. 48:5115). The selection criteria for hits was (1) increasedamount of ω-hydroxy fatty acids (ωOH FFA titer); and/or (2) increasedconversion of fatty acids to ω-hydroxy fatty acids.

Standard techniques known to those of skill in the art were used toprepare saturation libraries. Plasmids pEP125 and pLC81 (see Table 4,supra) were used to make the full saturation libraries. Three saturationlibraries were screened: For the first library pEP125 was transformedtogether with pNH305 into strain GLPH077, for the second library pLC81was transformed into BZ128, and for the third library pEP125 wastransformed together with pAS.033 into GLPH077Strain. The 1^(st) and2^(nd) library were screened in particular for improved variants inω-hydroxy dodecanoic acid formation and the 3^(rd) library was screenedin particular for improved variants in ω-hydroxy hexadecenoic acidformation. The libraries were screened using one of the standardprotocols described above. The improved variants are shown in Tables 5through 7 below (infra). In particular, variants of position 141 wereidentified multiple times and were found to be significantly improvedenzymes both for ω-hydroxy dodecanoic acid and ω-hydroxy hexadecenoicacid formation.

TABLE 5 Summary of improved variants from 1^(st) site saturation libraryof the catalytic domain of cyp153A(G307A)-Red450RhF. % C12:0 ω-OH Total% ω-OH ω-OH in Amino FFA FAS FFA FIOC C12:0 FIOC Acids Codons 1346.32236.6 60.2 1.33 83.1 1.08 V141Q GTG/CAG 1201.1 2149.3 55.9 1.23 84.11.10 D134G GAC/GGG 1106.2 2006.9 55.1 1.22 82 1.07 R40H AGG/CAC 1007.91839.7 54.8 1.21 86.1 1.12 V141I GTG/ATC 962.5 1791.2 53.7 1.19 81.11.06 K41V AAG/GTG 1228.6 2298.6 53.4 1.18 80.2 1.05 M419V ATG/GTC 1046.81958.5 53.4 1.18 80.1 1.05 V154A GTG/GCC 990.7 1865.4 53.1 1.17 84.91.11 D134G GAC/GGT 1203.1 2313.1 52 1.15 81.6 1.07 D134G GAC/GGG 908.71773.2 51.2 1.13 80.3 1.05 I11C ATT/TGC 1020.1 2057 49.6 1.09 81.4 1.06R205L CGC/TTG 1256 2688.4 46.7 1.03 72.6 0.95 L304W CTC/TGG 883.2 1960.845.3 1.00 76.6 1.00 FIOC: Fold improvement over control; control is bold

TABLE 6 Summary of improved variants from 2^(nd) site saturation libraryof the catalytic domain of cyp153A(G307A)-Red450RhF Total % C12:0 ω-OHTotal % ω-OH ω-OH in Mutation 1 Mutation 2 FFA FAS FFA FIOC C12:0 FASFIOC V415R 0 928.10 2880.10 32.23 1.85 33.29 1.96 V415R 0 941.13 2980.9731.58 1.81 32.98 1.94 V154A 0 694.63 2959.63 23.47 1.35 23.06 1.36 V154A0 716.00 2963.77 24.16 1.39 23.88 1.40 V154A 0 686.93 2926.97 23.47 1.3523.40 1.38 V141M E142Q 717.80 2873.73 24.98 1.44 28.51 1.68 V141I 0749.07 2971.23 25.21 1.45 31.96 1.88 V141I 0 778.87 2886.77 26.98 1.5534.27 2.02 V141I 0 754.67 2918.90 25.85 1.49 32.85 1.93 V141I R258Y672.13 2909.13 23.10 1.33 29.24 1.72 V141I 0 810.23 2912.67 27.83 1.6035.86 2.11 S233R 0 720.13 2838.00 25.37 1.46 30.82 1.81 S233R 0 746.202912.97 25.61 1.47 31.15 1.83 S233N 0 735.57 2905.40 25.33 1.46 25.771.52 S233N 0 698.80 2915.17 23.97 1.38 24.40 1.44 S233N 0 732.47 2949.9324.83 1.43 25.29 1.49 S233N 0 725.97 3018.60 24.05 1.38 24.76 1.46 R82DE271F 629.03 2914.83 21.58 1.24 20.90 1.23 R6F R178N 792.33 2845.1727.85 1.60 28.56 1.68 R6F V141I 833.13 2871.87 29.01 1.67 36.28 2.13R27L 0 742.57 2857.53 25.99 1.49 26.10 1.54 R178N 0 701.17 2983.60 23.501.35 24.98 1.47 Q129R 0 675.07 2847.37 23.71 1.36 27.97 1.65 Q129R 0812.23 3044.30 26.68 1.53 31.29 1.84 Q129R 0 660.53 2967.23 22.26 1.2826.24 1.54 P149R S157V 684.03 3011.80 22.71 1.31 23.04 1.36 P149R 0771.40 2959.70 26.06 1.50 26.12 1.54 P149R 0 731.10 2966.13 24.65 1.4224.75 1.46 P149R 0 757.97 3014.93 25.14 1.45 25.49 1.50 P149R 0 765.902963.50 25.84 1.49 26.16 1.54 P149R 0 734.30 2923.70 25.12 1.44 25.501.50 P149R 0 745.00 2993.83 24.88 1.43 25.47 1.50 P136T 0 724.53 2980.2024.31 1.40 24.97 1.47 P136T 0 729.37 3017.67 24.17 1.39 24.90 1.46 P136T0 678.33 2850.87 23.79 1.37 24.39 1.43 P136C 0 702.27 2947.23 23.83 1.3725.36 1.49 P136C 0 689.77 3069.63 22.47 1.29 24.01 1.41 N407A 0 731.503042.77 24.04 1.38 24.56 1.44 N407A 0 704.47 3015.93 23.36 1.34 23.751.40 M228R 0 344.60 2992.27 11.52 0.66 18.33 1.08 L168V 0 793.20 2938.2327.00 1.55 27.84 1.64 G161P 0 718.33 2938.47 24.45 1.41 24.28 1.43 G161A0 639.93 2943.40 21.74 1.25 21.65 1.27 G138F N407A 667.93 2825.43 23.641.36 26.09 1.53 F116R V415R 678.77 2854.97 23.78 1.37 24.14 1.42 E142R 0663.67 2925.83 22.68 1.30 22.86 1.34 E142R 0 628.03 2930.57 21.43 1.2321.62 1.27 E142R 0 639.23 2972.03 21.51 1.24 21.86 1.29 D153G 0 787.873018.90 26.13 1.50 26.94 1.58 D153G 0 746.20 3039.10 24.55 1.41 25.311.49 0 0 543.65 3117.75 17.44 1.00 17.04 1.00 FIOC: Fold improvementover control; control is bold

TABLE 7 Summary of improved variants from 3^(rd) site saturation libraryof the catalytic domain of cyp153A(G307A)-Red450RhF % C16:0 % C16:1 ω-OHTotal % ω-OH ω-OH in ω-OH in Amino FFA FAS FFA FIOC C16:0 FIOC C16:1FIOC Acids Codons 1298.5 2342.5 55.43 1.53 64.61 1.33 49.02 2.00 N309RAAC/CGG 1095.9 2374.3 46.16 1.28 58.36 1.20 34.41 1.40 V141G GTG/GGG1564 3448.1 45.36 1.25 62.78 1.29 32.88 1.34 L132T CTC/ACT 1092.9 2391.445.70 1.26 60.82 1.25 32.96 1.34 F144R TTC/AGG 1170.5 2529.6 46.27 1.2862.41 1.28 31.91 1.30 I131L ATT/TTG 1232.9 2685.8 45.90 1.27 55.17 1.1337.63 1.53 G308W GGC/TGG 931.1 2570.1 36.2 1.00 48.70 1.00 24.53 1.00FIOC: Fold improvement over control; control is bold

Example 3: Partial Site Saturation Libraries of the Reductase Domain ofcyp153A(G307A)-Red450RhF Fusion Protein

A partial saturation library (every 10^(th) amino acid was mutated) ofthe reductase domains of hybrid cyp153A-Red450RhF fusion protein, wasbuilt and screened for variants that showed improvements overcyp153A(V141I, A231T, G307A)-Red450RhF (SEQ ID NO: 32), a variantidentified in the site saturation mutagenesis library of the catalyticP450 cyp153A domain. The selection criteria for hits was (1) increasedamount of ω-hydroxy dodecanoic acid (ωOH FFA titer); and/or (2)increased conversion of dodecanoic acid to ω-hydroxy dodecanoic acid.

Standard techniques known to those of skill in the art were used toprepare saturation libraries. For the library, pLC81 harboringcyp153A(V141I, A231T, G307A)-Red450RhF was transformed into BZ128. Thelibrary was screened using one of the standard protocols describedabove. The improved variants are shown in Table 8. In particular thevariants A796V (SEQ ID: 42) and P666A were significantly improvedenzymes.

TABLE 8 Summary of improved variants from a partial saturation libraryof the reductase domain of cyp153A(V141I A231T G307A)-Red450RhF C12:0RhF ω-OH % ω-OH ω-OH in Mutation FFA FAS FFA FIOC C12:0 FAS FIOC P666K1012.1 2945.5 34.36 1.09 44.08 1.07 P666A 1575.9 2918.7 53.99 1.71 68.351.66 T516E 1150.4 2966.2 38.78 1.23 49.01 1.19 V696K 983.4 2955.4 33.271.05 43.02 1.05 0 950.3 3004.6 31.63 1.00 41.13 1.00 A796V 2458.0 3884.763.27 1.81 76.58 1.70 0 1363.7 3905.2 34.92 1.00 44.96 1.00 FIOC: Foldimprovement over control; control is bold

Example 4: Combinatorial Library of the Reductase Domain ofcyp153A(G307A)-Red450RhF Fusion Protein

Beneficial mutations identified in the partial saturation library of thereductase domain (Example 3) were the basis of a combination library tofurther improve cyp153A(G307A)-Red450RhF fusion protein. The selectioncriteria was (1) increased amount of ω-hydroxy dodecanoic acid (ωOH FFAtiter); and/or (2) increased conversion of dodecanoic acid to ω-hydroxydodecanoic acid.

The combination library was constructed in pLC81 harboringcyp153A(V141I, A231T, G307A)-Red450RhF (SEQ ID: 32) and transformed intoBZ128. Standard techniques known to those of skill in the art were usedto prepare combination libraries. The library was screened using one ofthe standard protocols described above. The improved variants are shownin Table 9 below.

TABLE 9 Summary of improved variants from a combination library of thereductase domain of cyp153A(V141I, A231T, G307A)-Red450RhF % C12:0 P450RhF ω-OH % ω-OH ω-OH in Mutation Mutation FFA FAS FFA FIOC C12:0 FASFIOC 141I, 231T T516G, P666M, A769V 851 983 86.8 1.29 88.3 1.23 141I,231T T516G, P666H, A769V 1557 2214 69.2 1.03 73.1 1.02 141I, 231T T516V,P666D, A769V 1491 1999 74.5 1.11 76.9 1.07 141I, 231T P666M, V696T916.88 1125 81.4 1.21 82.9 1.15 141I, 231T A769V 1528.33 2280 67.1 1.0071.8 1.00 FIOC: Fold improvement over control; control is bold

Example 5: Combinatorial Library of the Catalytic and Reductase Domainof cyp153A(G307A)-Red450RhF Fusion Protein

Beneficial mutations identified in the saturation libraries (Example 2and 3) were the basis of a combination library to further improvecyp153A(G307A)-Red450RhF fusion protein. The selection criteria was (1)increased amount of ω-hydroxy dodecanoic acid (ωOH FFA titer); and/or(2) increased conversion of dodecanoic acid to ω-hydroxy dodecanoicacid. The combination library was constructed in pLC81 and transformedinto BZ128. Standard techniques known to those of skill in the art wereused to prepare combination libraries. The library was screened usingone of the standard protocols described above. The best two improvedvariants are shown in Table 10.

TABLE 10 Best improved variants from a combinatorial library ofcyp153A(G307A)- Red450RhF SEQ ω-OH ω-OH C12:0 ω-OH Mutations ID FFA*FAS* FFA in C12:0 FAS R27I, R82D, 34 2290.3 3665.1 62.4% 74.1% V141M,R178N, N407A R27I, R82D, 44 3499.5 4154.9 84.5% 93.1% V141M, R178N,N407A, A796V *Titer (mg/L) after 48 h

Example 6: Site Saturation Mutagenesis of the Position 141 and 309 ofcyp153A(G307A, A796V)-Red450RhF

It was noticed that changes in position 141 influenced substratespecificity. Therefore, a site saturation mutagenesis of these twopositions were carried out in cyp153A(G307A, A796V)-Red450RhF. Theselection criteria for hits was (1) increased amount of ω-hydroxyhexadecenoic acid; and/or (2) increased conversion of hexadecenoic acidto ω-hydroxy hexadecenoic acid.

For the library, pEP146 harboring cyp153A(G307A A796V)-Red450RhF (SEQID: 38) was transformed into BZ128. Standard techniques known to thoseof skill in the art were used to prepare site saturation libraries. Thelibrary was screened using one of the standard protocols describedabove. The improved variants are shown in FIG. 2. In particular, thevariants with V141T (SEQ ID: 46) showed highest ω-hydroxy hexadecenoicacid titer and highest conversion from hexadecenoic acid.

Example 7: Saturation Libraries of cyp153A(G307A)-Red450RhF(A796V)Fusion Protein

A full saturation library of cyp153A-Red450RhF fusion protein, was builtand screened for variants that showed improvements overcyp153A(G307A)-Red450RhF(A796V) (i.e., the template polypeptide). G307A(i.e., an alanine residue replaced a glycine in position 307) and A796V(i.e., a valine residue replaced an alanine in position 796) arebeneficial mutations that improve ω-hydroxylase activity of cyp153A (seeabove). The selection criteria for hits was (1) an increased amount ofω-hydroxy fatty acids (ω-OH FFA titer); and/or (2) increased conversionof fatty acids to ω-hydroxy fatty acids.

Standard techniques known to those of skill in the art were used toprepare the saturation library. Plasmid pEP302 was used to make the fullsaturation library, which was a derivative of pEP146 (see table 4), inwhich the order of the genes was altered(fatA3-fadB-fadR-cyp153A(G307A)-Red450RhF(A796V)) and the last gene wasexpressed from a separate promoter. The library was transformed intostrain stNH1525. Briefly, the genome of base strain stNH1525 wasmanipulated as follows: the fadE (acyl-CoA dehydrogenase) gene wasdeleted and a synthetic fatty acid biosynthesis operon wasoverexpressed. In addition, the strain had previously been subjected totransposon as well as N-methyl-N′-nitro-N-nitrosoguanidine (NTG)mutagenesis and screening.

The libraries were screened using one of the standard protocolsdescribed above. The improved variants are shown in Table 11 below, inparticular, variants that significantly improved ω-hydroxy hexadecanoicacid and ω-hydroxy hexadecenoic acid formation.

TABLE 11 Summary of improved variants from the site saturation libraryof cyp153A(G307A)-Red450RhF(A976V) % C16:1 % C16:0 w-OH Total % w-OHw-OH in w-OH in Amino FFA FAS FFA FOIC C16:1 FOIC C16:0 FOIC acidsCodons 1201.0 1751.4 68.6 1.8 59.7 2.1 80.1 1.4 D747N AAC 1007.7 1733.358.1 1.6 50.6 1.9 79.0 1.4 Q12W TGG 793.2 1366.8 58.0 1.6 54.4 2.0 77.11.3 P327D GAT 955.6 1714.9 55.7 1.5 50.6 1.7 74.9 1.3 R14F TTC 678.71235.6 54.9 1.4 52.6 1.8 73.1 1.3 N61L TTG 855.8 1629.3 52.5 1.4 44.61.6 72.9 1.3 R27L TTG 911.6 1763.5 51.7 1.4 44.7 1.6 72.3 1.2 Q28M ATG858.1 1678.7 51.1 1.3 46.8 1.6 69.9 1.2 S13K AAG 1247.9 2458.5 50.8 1.344.3 1.6 67.8 1.2 V771F TTC 850.2 1686.3 50.4 1.3 42.1 1.5 71.6 1.2 Q12TACG 810.3 1615.4 50.2 1.3 46.8 1.6 65.0 1.1 K119R CGG 821.1 1639.9 50.11.3 43.7 1.6 70.1 1.2 D10Y TAC 807.4 1676.8 48.2 1.3 40.8 1.4 68.7 1.2Q12R AGG 722.5 1519.4 47.6 1.3 39.3 1.4 68.9 1.2 I11L TTG 748.9 1576.647.5 1.2 42.4 1.5 66.3 1.1 Q28T ACG 733.8 1546.2 47.5 1.3 38.7 1.4 68.91.2 A231Y TAC 1198.9 2528.5 47.4 1.2 39.9 1.4 67.5 1.2 P745R CGC/CGG769.8 1647.1 46.7 1.2 38.1 1.4 68.0 1.2 D9N AAT 1133.4 2469.6 45.9 1.238.1 1.4 65.6 1.1 T770G GGT 763.7 1672.4 45.7 1.2 39.0 1.4 63.2 1.1Y413R AGG 1146.2 2514.4 45.6 1.2 37.0 1.3 66.0 1.1 M784I ATC 729.81610.4 45.3 1.2 36.5 1.3 65.8 1.1 D9K AAG 1078.0 2390.8 45.1 1.2 35.71.3 66.7 1.1 E749L TTG 752.9 1682.8 44.7 1.2 38.1 1.3 63.7 1.1 S233L TTG940.3 2111.2 44.5 1.2 35.9 1.3 65.4 1.1 E757A GCG 1063.1 2405.5 44.2 1.235.5 1.3 65.0 1.1 L703G GGG 632.5 1434.9 44.1 1.2 37.5 1.3 61.5 1.1N309S TCT 755.7 1715.3 44.1 1.2 35.0 1.3 64.5 1.1 S140N AAC 1070.72441.3 43.9 1.2 37.6 1.3 60.9 1.0 L706E GAG 757.3 1753.4 43.2 1.2 33.71.2 65.2 1.1 I480G GGT 880.6 2044.0 43.1 1.2 33.6 1.2 65.3 1.1 G481I ATT989.8 2301.4 43.0 1.1 35.0 1.2 62.5 1.1 R719W TGG 1062.9 2478.7 42.9 1.135.7 1.3 61.1 1.0 L706S TCG 906.2 2116.3 42.8 1.1 34.8 1.3 62.5 1.1E557W TGG 734.0 1717.3 42.7 1.1 34.1 1.3 60.6 1.0 S157R CGG 651.4 1527.042.7 1.1 35.9 1.2 61.1 1.1 S233V GTC 710.4 1667.8 42.6 1.1 35.6 1.2 60.61.0 A231V GTA 663.4 1558.7 42.6 1.1 33.4 1.1 62.1 1.1 A164N AAC 664.31564.2 42.5 1.1 32.3 1.2 64.7 1.1 A244R CGG 711.2 1675.3 42.5 1.1 38.31.3 56.0 1.0 T302M ATG 1010.3 2381.5 42.4 1.1 34.1 1.2 62.4 1.1 P708STCG 1015.6 2394.2 42.4 1.1 32.3 1.1 64.9 1.1 N741G GGG 690.6 1630.5 42.41.1 33.3 1.2 63.0 1.1 P149G GGG 656.9 1552.9 42.3 1.1 34.8 1.3 60.9 1.0V154G GGC 905.2 2139.9 42.3 1.1 33.1 1.2 63.6 1.1 E557R CGG/AGG 946.62247.5 42.1 1.1 32.9 1.2 63.4 1.1 V710Q CAG 1159.1 2753.7 42.1 1.1 32.61.2 65.7 1.1 E567S TCC 640.8 1522.8 42.1 1.1 34.4 1.3 59.7 1.0 P149R AGG665.7 1587.4 41.9 1.1 33.0 1.2 61.8 1.1 N407G GGG 1026.9 2465.0 41.7 1.131.7 1.1 63.9 1.1 D544N AAC 941.4 2265.1 41.6 1.1 32.5 1.2 62.6 1.1D709L CTG 721.3 1747.4 41.3 1.1 35.1 1.2 58.3 1.0 G204V GTT 985.6 2393.741.2 1.1 31.7 1.1 62.7 1.1 V710C TGT 696.6 1694.0 41.1 1.1 32.3 1.2 61.51.1 R254G GGG 664.3 1616.1 41.1 1.1 32.8 1.1 61.1 1.1 P273M ATG 739.21801.0 41.0 1.1 33.8 1.2 59.9 1.0 F111A GCG 1042.8 2540.9 41.0 1.1 31.61.1 62.5 1.1 E749M ATG 681.8 1661.6 41.0 1.1 30.0 1.1 64.3 1.1 A231W TGG1017.8 2487.3 40.9 1.1 31.1 1.1 63.2 1.1 P546G GGG 719.6 1760.1 40.9 1.132.2 1.2 60.4 1.0 V162C TGC 950.8 2327.9 40.8 1.1 31.8 1.1 61.8 1.1A736V GTC 945.2 2314.2 40.8 1.1 34.2 1.2 58.5 1.0 L706H CAC 914.2 2241.140.8 1.1 31.4 1.1 62.3 1.1 V710R AGG 1055.3 2587.4 40.8 1.1 32.0 1.161.2 1.0 D707E GAG 883.4 2168.3 40.7 1.1 32.5 1.2 60.7 1.0 D527E GAG921.4 2266.5 40.7 1.1 32.9 1.2 60.3 1.0 P745K AAG 728.9 1804.5 40.4 1.132.2 1.1 60.3 1.0 E271D GAC 1031.5 2558.2 40.3 1.1 30.2 1.1 62.2 1.1E557R AGG 974.6 2429.2 40.1 1.1 30.6 1.1 61.8 1.1 D720V GTG 647.6 1616.540.1 1.1 30.2 1.1 61.2 1.1 P56Q CAG 934.9 2358.1 39.6 1.1 30.6 1.1 61.51.1 V648L TTG 938.8 2376.0 39.5 1.1 32.0 1.1 58.6 1.0 S649I ATC 672.11709.2 39.3 1.1 30.5 1.1 59.7 1.0 P477G GGG 878.5 2245.0 39.1 1.1 30.21.1 60.1 1.1 E591Q CAG 598.2 1582.9 37.8 1 28.4 1.0 57.8 1.0 FOIC: Foldimprovement over internal control; control is bold

As is apparent to one with skill in the art, various modifications andvariations of the above aspects and embodiments can be made withoutdeparting from the spirit and scope of this disclosure. Suchmodifications and variations are within the scope of this disclosure.

What is claimed is:
 1. A CYP153A-reductase hybrid fusion polypeptidevariant comprising at least 95% sequence identity to SEQ ID NO: 38 andhaving at least one mutation at an amino acid position corresponding toamino acid position 56, 61, 111, 140, 162, 164, 204, 244, 254, 273, 302,477, 480, 481, 527, 544, 546, 557, 567, 591, 648, 649, 706, 707, 708,709, 710, 719, 720, 736, and 741 of SEQ ID NO: 38, wherein the CYP153Areductase hybrid fusion polypeptide variant catalyzes conversion of afatty acid to an omega-hydroxylated fatty acid.
 2. The CYP153A-reductasehybrid fusion polypeptide variant of claim 1, wherein the mutation isselected from the group consisting of P56Q, N61L, F111A, 5140N, V162C,A164N, G204V, A244R, R254G, P273M, T302M, P477G, I480G, G481I, D527E,D544N, P546G, E557W, E557R, E567S, E591Q, V648L, S649I, L706H, L706E,L706S, D707E, P708S, D709L, V710C, V710R, V710Q, R719W, D720V, A736V,and N741G.
 3. A recombinant host cell comprising the CYP153A-reductasehybrid fusion polypeptide variant of claim
 1. 4. A cell culturecomprising the recombinant host cell of claim
 3. 5. A method forproducing an omega-hydroxylated fatty acid, the method comprising:culturing the recombinant host cell of claim 3 in a fermentation brothcomprising a carbon source.
 6. The method of claim 5, further comprisingisolating the omega-hydroxylated fatty acid.
 7. The method of claim 5,wherein the recombinant host cell further comprises an exogenousthioesterase polypeptide of EC 3.1.2.14 or EC 3.1.1.5.
 8. The method ofclaim 7, wherein the recombinant host cell produces a omega-hydroxylatedfatty acid with a titer that is at least 10% greater, at least 15%greater, at least 20% greater, at least 25% greater, or at least 30%greater than the titer of an omega-hydroxylated fatty acid produced by arecombinant host cell comprising a corresponding CYP153A-reductasehybrid fusion polypeptide of SEQ ID NO:
 38. 9. The method of claim 5,wherein the omega-hydroxylated fatty acid comprises one or more of aC₁₂, C₁₆ and a C_(16:1) omega-hydroxylated fatty acid.
 10. The method ofclaim 5, wherein the omega-hydroxylated fatty acid is producedintracellularly or extracellularly.